CN105916519A - Anti-siglec-8 antibodies and methods of use thereof - Google Patents

Abstract

The invention provides humanized anti-Siglec-8 antibodies and their use in treating and preventing eosinophil-mediated disorders and/or mast cell-mediated disorders, as well as compositions and kits comprising the humanized anti-Siglec-8 antibodies.

Description

Anti-Siglec-8 antibody and using method thereof

The intersection of related application is quoted

This application claims U.S. Provisional Patent Application No.61/913 of December in 2013 submission on the 9th, the priority of 891, incite somebody to action It is completely taken in herein by quoting.

The submission of sequence table on ASCII text file

The content of the following submission on ASCII text file is completely taken in herein by quoting: computer-reader form Sequence table (CRF) (file name: 701712000140SeqList.txt, record date: on December 9th, 2014, size: 115KB)。

Invention field

The present invention relates to anti-human Siglec-8 antibody and treatment or prevention by the cell-mediated disease expressing Siglec-8 Method.

Background of invention

Siglec (sialic acid binding domain-immunoglobulin sample agglutinin) is that the single pass transmembrane mainly found on the leukocytes is thin Cellular surface albumen, is characterised by they sialic specificitys to being attached to cell surface glycoconjugates.Siglec family contains There are at least 15 members found in mammal (Pillai et al., Annu Rev Immunol., 2012,30:357- 392).These members include sialoadhesin (Siglec-1), CD22 (Siglec-2), CD33 (Siglec-3), myelin Associated glycoprotein (Siglec-4), Siglec-5, OBBP1 (Siglec-6), AIRM1 (Siglec-7), SAF-2 (Siglec- , and CD329 (Siglec-9) 8).The member expressed in people but do not express in mice, Siglec-8 is initially conduct Identify the part discovery of the achievement of novel people's eosinocyte protein.Beyond being expressed by eosinocyte, it also by Mastocyte and basophil are expressed.Siglec-8 identifies a kind of sulfated glycan, i.e. 6 '-sulfo group-saliva acyl group Lewis X Or 6 '-sulfo group-saliva acyl group-N-acetyl group-S-lactose amine, and containing intracellular immunity receptor inhibition based on tyrosine Motif (ITIM) domain, it demonstrates suppression mastocyte function.

Together with mastocyte, eosinocyte can promote to play the inflammatory response of beneficial functionality effect, such as controls The infection at particular organization processed position.During inflammatory response, eosinophilic apoptosis can via survival promote cell because of The activity of son (such as IL-3 and GM-CSF) is suppressed.But, not over having activated of quickly removing of apoptosis addicted to eosin Cytosis may result in the release in inflamed sites already of the eosinocyte granule protein matter, and this understands damaging tissue and causes inflammation It is further exacerbated by.Several conditions has shown that is related with eosinophil activation, such as churg-Strauss syndrome, class wind Wet arthritis, and allergic asthma (Wechsler et al., J Allergy Clin Immunol., 2012,130 (3): 563-71).It is currently needed for controlling to relate to the activity (work of such as eosinocyte and mastocyte of the immunocyte of inflammation Property) therapy.

Previous research have turned out eosinocyte Siglec-8 with for Siglec-8 outside mitogenetic become Apoptosis (Nutku et al., Blood, 2003,336:918-24) is experienced when specific murine is antibody linked.These antibody are described in United States Patent (USP) No.8,207,305, United States Patent (USP) No.8,197,811, United States Patent (USP) No.7,871,612, and United States Patent (USP) No.7,557,191.It remains desirable, however, that exploitation resists with the humanization of high-affinity and specific recognition people Siglec-8 Siglec-8 antibody.The discovery tolerable of this type of anti-Siglec-8 antibody is developed by eosinocyte and/or the work of mastocyte Property mediation the treatment of disease.

By quoting, all references cited herein (is included patent application, patent publications, and science literary composition Offer) completely take in herein, just as clearly and individually pointing out to include each list of references by quoting.

Summary of the invention

Anti-Siglec-8 antibody provided herein (includes humanization anti-Siglec-8 antibody), comprises its compositions, and The method using it.

On the one hand, the humanized antibody of a kind of specific binding people Siglec-8 provided herein, wherein this humanization resists Body to the binding affinity of people Siglec-8 and/or binding affinity than antibody 2E2 and/or antibody 2C4 to people Siglec-8's Binding affinity and/or binding affinity want height.In some embodiments, this people Siglec-8 is dimer.Real at some Executing in scheme, this people Siglec-8 comprises the people's Siglec-8 extracellular domain being fused to immunoglobulin fc region.Some embodiment party In case, this Fc district is human IgG1 Fc district.In some embodiments, this Fc district is human IgG 4Fc district.In some embodiments, This people Siglec-8 comprises the aminoacid sequence of SEQ ID NO:74.

In some embodiments, this humanized antibody comprises variable region of heavy chain and variable region of light chain, and wherein this heavy chain can Becoming district and comprise the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, (ii) comprises the aminoacid of SEQ ID NO:62 The HVR-H2 of sequence, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:63；And/or wherein this light chain variable District comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, and (ii) comprises the aminoacid sequence of SEQ ID NO:65 The HVR-L2 of row, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:66.In some embodiments, this resists Body comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:6；And/or comprise selected from SEQ ID NO:16's or 21 The variable region of light chain of aminoacid sequence.In any embodiment in this article, this antibody can comprise human IgG Fc district Heavy chain Fc district.In further embodiment, this human IgG Fc district comprises human IgG1 or IgG4.In some embodiments In, this human IgG1 comprises the aminoacid sequence of SEQ ID NO:78.In some embodiments, this human IgG 4 comprises SEQ ID The aminoacid sequence of NO:79.In any embodiment in this article, this antibody can comprise the ammonia of SEQ ID NO:75 The heavy chain of base acid sequence；And/or comprise the light chain of aminoacid sequence selected from SEQ ID NO:76 or 77.In some embodiments In, this human IgG 4 comprises amino acid replacement S228P, and wherein amino acid residue is according to the EU index number in Kabat.At this In any embodiment in literary composition, this antibody can comprise the heavy chain of the aminoacid sequence of SEQ ID NO:87；And/or bag Light chain containing the aminoacid sequence of SEQ ID NO:76.In some embodiments, this antibody the most engineered is to improve Cytotoxicity (ADCC) activity of antibody dependent cellular mediation.In some embodiments, this antibody comprise two heavy chains and Wherein two heavy chains at least one of this antibody or two are non-fucosylations.

On the other hand, the humanized antibody of a kind of specific binding people Siglec-8 provided herein, wherein this antibody exists Hot transfer assay method (thermal shift assay) has the Tm of at least about 70 DEG C at least about 72 DEG C.Implement at some In scheme, this antibody has about 70 DEG C in hot transfer assay method, about 71 DEG C, or the Tm of about 72 DEG C.In some embodiments, This antibody has Tm same or higher compared with chimeric 2C4 antibody.In some embodiments, this antibody has and has bag The heavy chain of the aminoacid sequence containing SEQ ID NO:84 and comprise the antibody phase of light chain of aminoacid sequence of SEQ ID NO:85 Than same or higher Tm.

In some embodiments, this antibody comprises variable region of heavy chain and variable region of light chain, wherein this variable region of heavy chain bag Comprise the HVR-H1 of the aminoacid sequence of SEQ ID NO:61 containing (i), (ii) comprises the aminoacid sequence of SEQ ID NO:62 HVR-H2, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:63；And/or wherein this variable region of light chain comprises I () comprises the HVR-L1 of the aminoacid sequence of SEQ ID NO:64, (ii) comprises the aminoacid sequence of SEQ ID NO:65 HVR-L2, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:66.In some embodiments, this antibody bag Variable region of heavy chain containing the aminoacid sequence comprising SEQ ID NO:6；And/or comprise the amino selected from SEQ ID NO:16 or 21 The variable region of light chain of acid sequence.In any embodiment in this article, this antibody can comprise the weight in human IgG Fc district Chain Fc district.In further embodiment, this human IgG Fc district comprises human IgG1 or IgG4.In some embodiments, should Human IgG1 comprises the aminoacid sequence of SEQ ID NO:78.In some embodiments, this human IgG 4 comprises SEQ ID NO:79 Aminoacid sequence.In any embodiment in this article, this antibody can comprise the aminoacid of SEQ ID NO:75 The heavy chain of sequence；And/or comprise the light chain of aminoacid sequence selected from SEQ ID NO:76 or 77.In some embodiments, This human IgG 4 comprises amino acid replacement S228P, and wherein amino acid residue is according to the EU index number in Kabat.Herein In any embodiment in, this antibody can comprise the heavy chain of the aminoacid sequence of SEQ ID NO:87；And/or comprise The light chain of the aminoacid sequence of SEQ ID NO:76.In some embodiments, this antibody the most engineered is anti-to improve Cytotoxicity (ADCC) activity of body dependent cell mediation.In some embodiments, this antibody heavy chain at least one or Article two, it is non-fucosylation.

Yet another aspect, the humanized antibody of a kind of specific binding people Siglec-8 provided herein, wherein this resists Body comprises variable region of heavy chain and variable region of light chain, and wherein this variable region of heavy chain comprises the aminoacid that (i) comprises SEQ ID NO:61 The HVR-H1 of sequence, (ii) comprises the HVR-H2 of the aminoacid sequence of SEQ ID NO:62, and (iii) comprises selected from SEQ ID The HVR-H3 of the aminoacid sequence of NO:63 and 67-70；And/or wherein this variable region of light chain comprises (i) and comprises SEQ ID NO:64 The HVR-L1 of aminoacid sequence, (ii) comprises the HVR-L2 of the aminoacid sequence of SEQ ID NO:65, and (iii) comprises SEQ The HVR-L3 of the aminoacid sequence of ID NO:66 or 71.In some embodiments, this antibody comprises selected from SEQ ID The variable region of heavy chain of the aminoacid sequence of NO:11-14；And/or comprise the light of the aminoacid sequence selected from SEQ ID NO:23-24 Chain variable region.In some embodiments, this variable region of heavy chain comprises aminoacid sequence that (i) comprise SEQ ID NO:61 HVR-H1, (ii) comprises the HVR-H2 of the aminoacid sequence of SEQ ID NO:62, and (iii) comprises selected from SEQ ID NO:63's The HVR-H3 of aminoacid sequence；And/or this variable region of light chain comprises the HVR-that (i) comprises the aminoacid sequence of SEQ ID NO:64 L1, (ii) comprises the HVR-L2 of the aminoacid sequence of SEQ ID NO:65, and (iii) comprises the aminoacid sequence of SEQ ID NO:66 The HVR-L3 of row.In some embodiments, the heavy chain that this antibody comprises selected from the aminoacid sequence of SEQ ID NO:6 can Become district；And/or comprise the variable region of light chain of aminoacid sequence selected from SEQ ID NO:16 or 21.Any enforcement in this article In scheme, this antibody can comprise the heavy chain Fc district in human IgG Fc district.In further embodiment, this human IgG Fc District comprises human IgG1 or IgG4.In some embodiments, this human IgG1 comprises the aminoacid sequence of SEQ ID NO:78.One In a little embodiments, this human IgG 4 comprises the aminoacid sequence of SEQ ID NO:79.In some embodiments, this human IgG 4 wraps Containing amino acid replacement S228P, wherein amino acid residue is according to the EU index number in Kabat.In some embodiments, This antibody the most engineered is to improve cytotoxicity (ADCC) activity of antibody dependent cellular mediation.Some embodiment party In case, the heavy chain at least one of this antibody or two are non-fucosylations.

Yet another aspect, the humanized antibody of a kind of specific binding people Siglec-8 provided herein, wherein this resists Body comprises the variable region of heavy chain of the aminoacid sequence selected from SEQ ID NO:2-14；And/or comprise selected from SEQ ID NO: The variable region of light chain of the aminoacid sequence of 16-24.In some embodiments, this antibody the most engineered is to improve antibody Cytotoxicity (ADCC) activity of dependent cell mediation.In some embodiments, the heavy chain at least one or two of this antibody Bar is non-fucosylation.

Yet another aspect, the humanized antibody of a kind of specific binding people Siglec-8 provided herein, wherein this resists Body comprises the variable region of heavy chain of the aminoacid sequence selected from SEQ ID NO:2-10；And/or comprise selected from SEQ ID NO: The variable region of light chain of the aminoacid sequence of 16-22.In some embodiments, this antibody the most engineered is to improve antibody Cytotoxicity (ADCC) activity of dependent cell mediation.In some embodiments, the heavy chain at least one or two of this antibody Bar is non-fucosylation.

On the other hand, the humanized antibody of a kind of specific binding people Siglec-8 provided herein, wherein this antibody bag Containing variable region of heavy chain and variable region of light chain, wherein (a) this variable region of heavy chain comprises: (1) comprises selected from SEQ ID NO:26-29's The HC-FR1 of aminoacid sequence；(2) HVR-H1 of the aminoacid sequence of SEQ ID NO:61 is comprised；(3) comprise selected from SEQ ID The HC-FR2 of the aminoacid sequence of NO:31-36；(4) HVR-H2 of the aminoacid sequence of SEQ ID NO:62 is comprised；(5) comprise HC-FR3 selected from the aminoacid sequence of SEQ ID NO:38-43；(6) HVR-of the aminoacid sequence of SEQ ID NO:63 is comprised H3；(7) comprise the HC-FR4 of aminoacid sequence selected from SEQ ID NO:45-46, and/or (b) this variable region of light chain comprise: (1) HC-FR1 of aminoacid sequence selected from SEQ ID NO:48-49 is comprised；(2) the aminoacid sequence of SEQ ID NO:64 is comprised The HVR-H1 of row；(3) HC-FR2 of aminoacid sequence selected from SEQ ID NO:51-53 is comprised；(4) SEQ ID NO:65 is comprised The HVR-H2 of aminoacid sequence；(5) HC-FR3 of aminoacid sequence selected from SEQ ID NO:55-58 is comprised；(6) comprise The HVR-H3 of the aminoacid sequence of SEQ ID NO:66；(7) HC-FR4 of the aminoacid sequence of SEQ ID NO:60 is comprised.? In some embodiments, this antibody the most engineered is lived with the cytotoxicity (ADCC) improving antibody dependent cellular mediation Property.In some embodiments, the heavy chain at least one of this antibody or two are non-fucosylations.

Yet another aspect, the antibody of a kind of separation provided herein, it combines people Siglec-8 and passes through ADCC activity Kill the mastocyte expressing Siglec-8.In some embodiments, this antibody kills the fertilizer expressing Siglec-8 in vitro Maxicell (is measured in the cell culture assays described the most in example 2).In some embodiments, this antibody When administering therapeutic effective dose, in experimenter, abatement expresses the mastocyte of Siglec-8.A further embodiment In, compared with baseline values before treatment, this antibody abatement in the sample that this experimenter obtains at least about 20% (the most extremely The mastocyte of expression Siglec-8 less).In any embodiment in this article, this sample can be tissue sample or life Thing fluid sample.In some embodiments, this tissue sample is one or more selected from lower group: skin, lung, bone marrow, And nasal polyp.In some embodiments, this biological fluid sample is one or more selected from lower group: blood, bronchus Bronchoalveolar lavage fluid, and Nasal lavage fluid.In any embodiment in this article, can this antibody engineered to improve antibody Cytotoxicity (ADCC) activity of dependent cell mediation.In some embodiments, the heavy chain at least one or two of this antibody Bar is non-fucosylation.In further embodiment, can have α 1,6-fucosyltransferase (Fut8) strikes The cell line removed generates this antibody.In some further embodiments, can be at process LAN β Isosorbide-5-Nitrae-N-acetyl-glucosamine The cell line of based transferase (acetylglycosminyltransferase) III (GnT-III) generates this antibody.Entering one In the embodiment of step, this cell line other process LAN Gorky μ-mannosidase II (ManII).Any reality in this article Executing in scheme, this antibody can comprise at least one place in Fc district and improve the amino acid replacement of ADCC activity.In this article appoint In what embodiment, this antibody can be humanized antibody, chimeric antibody or people's antibody.In some embodiments, this antibody It is human IgG1's antibody.In some embodiments, this antibody is murine antibody.In any embodiment in this article, this antibody Can comprise variable region of heavy chain, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:88, and (ii) comprises SEQ The HVR-H2 of the aminoacid sequence of ID NO:91, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:94；With/ Or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:97, and (ii) comprises SEQ ID NO: The HVR-L2 of the aminoacid sequence of 100, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:103.Enter at one In the embodiment of one step, this antibody comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:106；And/or bag Variable region of light chain containing the aminoacid sequence of SEQ ID NO:109.In any embodiment in this article, this antibody can wrap Containing variable region of heavy chain, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:89, and (ii) comprises SEQ ID NO: The HVR-H2 of the aminoacid sequence of 92, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:95；And/or light chain Variable region, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:98, and (ii) comprises SEQ ID NO:101's The HVR-L2 of aminoacid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:104.At one further Embodiment in, this antibody comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:107；And/or comprise SEQ The variable region of light chain of the aminoacid sequence of ID NO:110.In any embodiment in this article, this antibody can comprise heavy chain Variable region, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:90, and (ii) comprises the ammonia of SEQ ID NO:93 The HVR-H2 of base acid sequence, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:96；And/or variable region of light chain, It comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:99, and (ii) comprises the aminoacid sequence of SEQ ID NO:102 The HVR-L2 of row, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:105.A further embodiment party In case, this antibody comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:108；And/or comprise SEQ ID NO: The variable region of light chain of the aminoacid sequence of 111.In any embodiment in this article, this antibody can comprise variable region of heavy chain And variable region of light chain, wherein this variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, (ii) Comprise the HVR-H2 of the aminoacid sequence of SEQ ID NO:62, and (iii) comprises the aminoacid sequence of SEQ ID NO:63 HVR-H3；And/or wherein this variable region of light chain comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, (ii) Comprise the HVR-L2 of the aminoacid sequence of SEQ ID NO:65, and (iii) comprises the aminoacid sequence of SEQ ID NO:66 HVR-L3.In any embodiment in this article, this antibody can comprise variable region of heavy chain and variable region of light chain, and wherein this is heavy Chain variable region comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises the ammonia of SEQ ID NO:62 The HVR-H2 of base acid sequence, and (iii) comprise the HVR-H3 of the aminoacid sequence selected from SEQ ID NO:67-70；And/or wherein This variable region of light chain comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, and (ii) comprises SEQ ID NO:65 The HVR-L2 of aminoacid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:71.In this article appoint In what embodiment, this experimenter can be people.

Still further aspect, a kind of antibody provided herein, it combines people Siglec-8 and non-human primates Siglec-8. In any embodiment in this article, this antibody can comprise variable region of heavy chain, and it comprises (i) and comprises SEQ ID NO:89's The HVR-H1 of aminoacid sequence, (ii) comprises the HVR-H2 of the aminoacid sequence of SEQ ID NO:92, and (iii) comprises SEQ ID The HVR-H3 of the aminoacid sequence of NO:95；And/or variable region of light chain, it comprises the aminoacid sequence that (i) comprises SEQ ID NO:98 The HVR-L1 of row, (ii) comprises the HVR-L2 of the aminoacid sequence of SEQ ID NO:101, and (iii) comprises SEQ ID NO:104 The HVR-L3 of aminoacid sequence.In a further embodiment, this antibody comprises the ammonia of SEQ ID NO:107 The variable region of heavy chain of base acid sequence；And/or comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:110.In this article Any embodiment in, this antibody can comprise variable region of heavy chain, and it comprises the aminoacid sequence that (i) comprises SEQ ID NO:90 The HVR-H1 of row, (ii) comprises the HVR-H2 of the aminoacid sequence of SEQ ID NO:93, and (iii) comprises SEQ ID NO:96's The HVR-H3 of aminoacid sequence；And/or variable region of light chain, it comprises aminoacid sequence that (i) comprise SEQ ID NO:99 HVR-L1, (ii) comprises the HVR-L2 of the aminoacid sequence of SEQ ID NO:102, and (iii) comprises the ammonia of SEQ ID NO:105 The HVR-L3 of base acid sequence.In a further embodiment, this antibody comprises the aminoacid of SEQ ID NO:108 The variable region of heavy chain of sequence；And/or comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:111.In this article appoint In what embodiment, this antibody (can such as comprise the aminoacid sequence of SEQ ID NO:112 in conjunction with people's Siglec-8 domain 1 Row domain 1) in epi-position.In any embodiment in this article, this antibody can be in conjunction with people's Siglec-8 domain 3 Epi-position in (such as comprising the domain 3 of the aminoacid sequence of SEQ ID NO:114).Any embodiment in this article In, this antibody (can such as comprise the domain of the aminoacid sequence of SEQ ID NO:113 in conjunction with people's Siglec-8 domain 2 2) epi-position in.In any embodiment in this article, this antibody can be humanized antibody, chimeric antibody or people's antibody. In some embodiments, this antibody is murine antibody.In some embodiments, this antibody be IgG1 or IgG4 antibody (such as Human IgG1 or IgG4).

On the other hand, one provided herein anti-human Siglec-8 antibody, it combines the amino comprising SEQ ID NO:116 Acid fusion protein but do not combine the amino acid whose fusion protein comprising SEQ ID NO:115.In some embodiments, herein Amino acid whose fusion protein that described antibodies comprises SEQ ID NO:117 but do not combine the ammonia comprising SEQ ID NO:115 The fusion protein of base acid.In some embodiments, antibodies described herein comprises amino acid whose the melting of SEQ ID NO:117 Hop protein but do not combine the amino acid whose fusion protein comprising SEQ ID NO:116.In some embodiments in this article, should Antibody can comprise variable region of heavy chain, and it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:88, and (ii) comprises The HVR-H2 of the aminoacid sequence of SEQ ID NO:91, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:94； And/or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:97, and (ii) comprises SEQ ID The HVR-L2 of the aminoacid sequence of NO:100, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:103.One In individual further embodiment, this antibody comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:106；With/ Or comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:109.In some embodiments in this article, this antibody can To comprise variable region of heavy chain, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:89, and (ii) comprises SEQ ID The HVR-H2 of the aminoacid sequence of NO:92, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:95；And/or it is light Chain variable region, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:98, and (ii) comprises SEQ ID NO:101 The HVR-L2 of aminoacid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:104.One is entered at one In the embodiment of step, this antibody comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:107；And/or comprise The variable region of light chain of the aminoacid sequence of SEQ ID NO:110.In some embodiments in this article, this antibody can comprise Variable region of heavy chain, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:90, and (ii) comprises SEQ ID NO:93 The HVR-H2 of aminoacid sequence, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:96；And/or light chain can Becoming district, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:99, and (ii) comprises the ammonia of SEQ ID NO:102 The HVR-L2 of base acid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:105.At one further In embodiment, this antibody comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:108；And/or comprise SEQ The variable region of light chain of the aminoacid sequence of ID NO:111.

On the other hand, the humanized antibody of a kind of combination people Siglec-8 provided herein, wherein cut down the people activated Eosinophilic EC₅₀Less than antibody 2E2 or the 2C4 EC to people Siglec-8₅₀.In some embodiments, this humanization resists The EC of body₅₀It is antibody 2E2 or the 2C4 EC to people Siglec-8₅₀About 85% or less.In some embodiments, this people source Change the EC of antibody₅₀It is antibody 2E2 or the 2C4 EC to people Siglec-8₅₀About 85%, about 80%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10% or About 5% or less.In any embodiment in this article, this humanized antibody can comprise variable region of heavy chain and light chain variable District, wherein this variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises SEQ ID The HVR-H2 of the aminoacid sequence of NO:62, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:63；And/or its In this variable region of light chain comprise the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, (ii) comprises SEQ ID NO: The HVR-L2 of the aminoacid sequence of 65, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:66.In this article In any embodiment, this humanized antibody can comprise the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:6； And/or comprise the variable region of light chain of aminoacid sequence selected from SEQ ID NO:16 or 21.In some embodiments, this people source Changing antibody and comprise variable region of heavy chain and variable region of light chain, wherein this variable region of heavy chain comprises the ammonia that (i) comprises SEQ ID NO:61 The HVR-H1 of base acid sequence, (ii) comprises the HVR-H2 of the aminoacid sequence of SEQ ID NO:62, and (iii) comprises selected from SEQ The HVR-H3 of the aminoacid sequence of ID NO:67-70；And/or wherein this variable region of light chain comprises (i) and comprises SEQ ID NO:64 The HVR-L1 of aminoacid sequence, (ii) comprises the HVR-L2 of the aminoacid sequence of SEQ ID NO:65, and (iii) comprises SEQ The HVR-L3 of the aminoacid sequence of ID NO:71.In any embodiment in this article, this humanized antibody can comprise bag Variable region of heavy chain containing the aminoacid sequence selected from SEQ ID NO:2-14；And/or comprise the ammonia selected from SEQ ID NO:16-24 The variable region of light chain of base acid sequence.In any embodiment in this article, this antibody can comprise variable region of heavy chain, and it comprises I () comprises the HVR-H1 of the aminoacid sequence of SEQ ID NO:88, (ii) comprises the aminoacid sequence of SEQ ID NO:91 HVR-H2, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:94；And/or variable region of light chain, it comprises (i) Comprising the HVR-L1 of the aminoacid sequence of SEQ ID NO:97, (ii) comprises the HVR-of the aminoacid sequence of SEQ ID NO:100 L2, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:103.In any embodiment in this article, this resists Body can comprise variable region of heavy chain, and it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:89, and (ii) comprises The HVR-H2 of the aminoacid sequence of SEQ ID NO:92, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:95； And/or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:98, and (ii) comprises SEQ ID The HVR-L2 of the aminoacid sequence of NO:101, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:104.At this In any embodiment in literary composition, this antibody can comprise variable region of heavy chain, and it comprises the amino that (i) comprises SEQ ID NO:90 The HVR-H1 of acid sequence, (ii) comprises the HVR-H2 of the aminoacid sequence of SEQ ID NO:93, and (iii) comprises SEQ ID NO: The HVR-H3 of the aminoacid sequence of 96；And/or variable region of light chain, it comprises the aminoacid sequence that (i) comprises SEQ ID NO:99 HVR-L1, (ii) comprises the HVR-L2 of the aminoacid sequence of SEQ ID NO:102, and (iii) comprises SEQ ID NO:105's The HVR-L3 of aminoacid sequence.In any embodiment in this article, this antibody can comprise the weight in human IgG Fc district Chain Fc district.In further embodiment, this human IgG Fc district comprises human IgG1 or IgG4.In some embodiments, should Human IgG1 comprises the aminoacid sequence of SEQ ID NO:78.In some embodiments, this human IgG 4 comprises SEQ ID NO:79 Aminoacid sequence.In any embodiment in this article, this antibody can comprise the aminoacid of SEQ ID NO:75 The heavy chain of sequence；And/or comprise the light chain of aminoacid sequence selected from SEQ ID NO:76 or 77.In some embodiments, This human IgG 4 comprises amino acid replacement S228P, and wherein amino acid residue is according to the EU index number in Kabat.Herein In any embodiment in, this antibody can comprise the heavy chain of the aminoacid sequence of SEQ ID NO:87；And/or comprise The light chain of the aminoacid sequence of SEQ ID NO:76.

On the other hand, a kind of nucleic acid provided herein, its coding above with any antibody described herein.Also has the opposing party Face, a kind of carrier provided herein, it comprises nucleic acid described herein.In one embodiment, this carrier is expression vector.Also Having on the other hand, a kind of host cell provided herein, it comprises nucleic acid described herein.In some embodiments, this host Cell is expressed and generates this antibody.

On the other hand, a kind of method generating antibody provided herein, it is cultivated under conditions of being included in this antibody of generation Comprise the host cell of one or more nucleic acid encoding antibody described herein.In some embodiments, the method is further The antibody generated by this host cell including recovery.The anti-Siglec-8 antibody that by the method generated is also provided herein.This Literary composition also provides for the Fab of anti-Siglec-8 antibody described herein.

On the other hand, a kind of pharmaceutical composition provided herein, it comprises ties with antibody described herein or its antigen above Close fragment and pharmaceutically acceptable supporting agent.

On the other hand, a kind of antibody comprising specific binding people Siglec-8 provided herein or the combination of its fragment Thing, wherein this antibody comprises Fc district and is connected to the carbohydrate chain that the N-glucosides in this Fc district connects, wherein in said composition The carbohydrate chain that this N-glucosides less than 50% connects contains fucosyl residues.In some embodiments, substantially free of The carbohydrate chain that one this N-glucosides connects contains fucosyl residues.In some embodiments, this antibody is that humanization resists Body, chimeric antibody or people's antibody.In some embodiments, this antibody comprises variable region of heavy chain and variable region of light chain, wherein should Variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises SEQ ID NO:62's The HVR-H2 of aminoacid sequence, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:63；And/or wherein this is light Chain variable region comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, and (ii) comprises the ammonia of SEQ ID NO:65 The HVR-L2 of base acid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:66.In some embodiments In, this antibody comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:2-10；And/or comprise SEQ ID NO: The variable region of light chain of the aminoacid sequence of 16-22.In some embodiments, this antibody comprises variable region of heavy chain and light chain variable District, wherein this variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises SEQ ID The HVR-H2 of the aminoacid sequence of NO:62, and (iii) comprise the HVR-H3 of the aminoacid sequence selected from SEQ ID NO:67-70； And/or wherein this variable region of light chain comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, (ii) comprises SEQ The HVR-L2 of the aminoacid sequence of ID NO:65, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:71.One In a little embodiments, this antibody comprises the variable region of heavy chain of the aminoacid sequence selected from SEQ ID NO:11-14；And/or Comprise the variable region of light chain of aminoacid sequence selected from SEQ ID NO:23-24.In some embodiments, this antibody comprises bag Variable region of heavy chain containing the aminoacid sequence selected from SEQ ID NO:2-14；And/or comprise the ammonia selected from SEQ ID NO:16-24 The variable region of light chain of base acid sequence.In any embodiment in this article, said composition can comprise pharmacy further and can connect By supporting agent.In any embodiment in this article, the binding affinity of this antibody on human Siglec-8 and/or binding affinity Can be higher to binding affinity and/or the binding affinity of people Siglec-8 than antibody 2E2 or 2C4.In this article any In embodiment, this antibody can have the Tm of at least about 70 DEG C at least about 72 DEG C in hot transfer assay method.Real at some Executing in scheme, this antibody has about 70 DEG C, about 71 DEG C, or the Tm of about 72 DEG C.In some embodiments, this antibody has with embedding Close 2C4 antibody and compare same or higher Tm.In some embodiments, this antibody has and comprises SEQ ID NO:84 with having The heavy chain of aminoacid sequence compare same or higher with the antibody of the light chain of the aminoacid sequence comprising SEQ ID NO:85 Tm。

On the other hand, provided herein a kind of treat in experimenter or prevent by express Siglec-8 cell-mediated The method of disease, antibody described herein that the method includes this experimenter uses effective dose or its Fab or herein Described compositions.In some embodiments, this disease is Eosinophils mediate disease.In some embodiments, this disease Disease is mast cell mediated disease.In some embodiments, this disease is selected from lower group: asthma, allergic rhinitis, nasal polyp Disease, atopic dermatitis, chronic urticaria, Mastocytosis, eosinophilic leukemia, and eosinophilia is comprehensive Levy.In some embodiments, this disease is selected from lower group: few granulocytic asthma, acute or chronic airways hypersensitivity, addicted to daybreak Fragility of erythrocytes esophagitis, churg-Strauss syndrome, the inflammation relevant with cytokine, have with the cell expressing Siglec-8 The inflammation closed, relevant with the cell expressing Siglec-8 is pernicious, physical urticaria, cold urticaria, repressive Herba Urticae Cannabinae Rash, bullous pemphigoid, food anaphylaxis, and allergic bronchopulmonary aspergillosis (ABPA).In some embodiments, this resists One or more symptoms of body suppression atopic reaction.In some embodiments, this atopic reaction is that I type hypersensitivity is anti- Should.In any embodiment in this article, this experimenter can suffer from by suction-type corticosteroid, and fugitive β 2 is exciting Agent, long-acting β2agonists, or the asthma that a combination thereof is the most appropriately controlled.

On the other hand, a kind of method that abatement expresses the mastocyte of Siglec-8 in experimenter provided herein, its Including the antibody of the combination people Siglec-8 that this experimenter uses effective dose, wherein this antibody kills expression by ADCC activity The mastocyte of Siglec-8.In some embodiments, this antibody kills the mastocyte (example expressing Siglec-8 in vitro Cell culture assays as described in example 2 is measured).In some embodiments, with baseline before treatment Level is compared, this antibody abatement mastocyte of the expression Siglec-8 of at least about 20% in the sample that this experimenter obtains. In any embodiment in this article, this sample can be tissue sample or biological fluid sample.In some embodiments In, this tissue is one or more selected from lower group: skin, lung, bone marrow, and nasal polyp.In some embodiments, this biology Learning fluid sample is one or more selected from lower group: blood, bronchoalveolar lavage fluid, and Nasal lavage fluid.In this article In any embodiment, can live with the cytotoxicity (ADCC) improving antibody dependent cellular mediation by this antibody engineered Property.In any embodiment in this article, this antibody can comprise two heavy chains and wherein this antibody two heavy chains at least Article one, or two be non-fucosylation.In further embodiment, this antibody can have a α 1,6-fucose The cell line that based transferase (Fut8) knocks out generates.In some further embodiments, this antibody can be at process LAN β The cell line of 1,4-N-acetylglucsoaminyltransferase III (GnT-III) generates.In further embodiment, this is thin Born of the same parents are other process LAN Gorky μ-mannosidase II (ManII).In any embodiment in this article, this antibody is permissible In Fc district, comprise at least one place improve the amino acid replacement of ADCC activity.In any embodiment in this article, this antibody Can be humanized antibody, chimeric antibody or people's antibody.In some embodiments, this antibody is human IgG1's antibody.Herein In any embodiment in, this antibody can comprise variable region of heavy chain, and it comprises the aminoacid that (i) comprises SEQ ID NO:88 The HVR-H1 of sequence, (ii) comprises the HVR-H2 of the aminoacid sequence of SEQ ID NO:91, and (iii) comprises SEQ ID NO:94 The HVR-H3 of aminoacid sequence；And/or variable region of light chain, it comprises aminoacid sequence that (i) comprise SEQ ID NO:97 HVR-L1, (ii) comprises the HVR-L2 of the aminoacid sequence of SEQ ID NO:100, and (iii) comprises the ammonia of SEQ ID NO:103 The HVR-L3 of base acid sequence.In a further embodiment, this antibody comprises the aminoacid of SEQ ID NO:106 The variable region of heavy chain of sequence；And/or comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:109.In this article appoint In what embodiment, this antibody can comprise variable region of heavy chain, and it comprises aminoacid sequence that (i) comprise SEQ ID NO:89 HVR-H1, (ii) comprises the HVR-H2 of the aminoacid sequence of SEQ ID NO:92, and (iii) comprises the amino of SEQ ID NO:95 The HVR-H3 of acid sequence；And/or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:98, (ii) comprise the HVR-L2 of the aminoacid sequence of SEQ ID NO:101, and (iii) comprises the aminoacid sequence of SEQ ID NO:104 The HVR-L3 of row.In a further embodiment, this antibody comprises the aminoacid sequence of SEQ ID NO:107 Variable region of heavy chain；And/or comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:110.Any enforcement in this article In scheme, this antibody can comprise variable region of heavy chain, and it comprises the HVR-that (i) comprises the aminoacid sequence of SEQ ID NO:90 H1, (ii) comprises the HVR-H2 of the aminoacid sequence of SEQ ID NO:93, and (iii) comprises the aminoacid sequence of SEQ ID NO:96 The HVR-H3 of row；And/or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:99, (ii) Comprise the HVR-L2 of the aminoacid sequence of SEQ ID NO:102, and (iii) comprises the aminoacid sequence of SEQ ID NO:105 HVR-L3.In a further embodiment, this antibody comprises the heavy chain of the aminoacid sequence of SEQ ID NO:108 Variable region；And/or comprise the variable region of light chain of the aminoacid sequence of SEQ ID NO:111.Any embodiment in this article In, this antibody can comprise variable region of heavy chain and variable region of light chain, and wherein this variable region of heavy chain comprises (i) and comprises SEQ ID NO: The HVR-H1 of the aminoacid sequence of 61, (ii) comprises the HVR-H2 of the aminoacid sequence of SEQ ID NO:62, and (iii) comprises The HVR-H3 of the aminoacid sequence of SEQ ID NO:63；And/or wherein this variable region of light chain comprises (i) and comprises SEQ ID NO:64 The HVR-L1 of aminoacid sequence, (ii) comprises the HVR-L2 of the aminoacid sequence of SEQ ID NO:65, and (iii) comprises SEQ The HVR-L3 of the aminoacid sequence of ID NO:66.In any embodiment in this article, this antibody can comprise weight chain variable District and variable region of light chain, wherein this variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, (ii) comprise the HVR-H2 of the aminoacid sequence of SEQ ID NO:62, and (iii) comprises the amino selected from SEQ ID NO:67-70 The HVR-H3 of acid sequence；And/or wherein this variable region of light chain comprises the HVR-that (i) comprises the aminoacid sequence of SEQ ID NO:64 L1, (ii) comprises the HVR-L2 of the aminoacid sequence of SEQ ID NO:65, and (iii) comprises the aminoacid sequence of SEQ ID NO:71 The HVR-L3 of row.In any embodiment in this article, this experimenter has by the cell-mediated disease expressing Siglec-8 Sick.In some embodiments, this disease is selected from lower group: asthma, allergic rhinitis, nasal polyp, atopic dermatitis, chronic nettle Measles, Mastocytosis, eosinophilic leukemia, and eosinophilia's syndrome.In some embodiments, This disease is selected from lower group: few granulocytic asthma (pauci granulocytic asthma), acute or chronic air flue hypersensitization Property, eosinophilic esophagitis, churg-Strauss syndrome, the inflammation relevant with cytokine, with expression Siglec-8's The inflammation that cell is relevant, relevant with the cell expressing Siglec-8 is pernicious, physical urticaria, cold urticaria, compressing Property urticaria, bullous pemphigoid, food anaphylaxis, and allergic bronchopulmonary aspergillosis (ABPA).In some embodiments In, one or more symptoms of this antibody suppression atopic reaction.In some embodiments, this atopic reaction is that I type surpasses Sensitive response.In any embodiment in this article, this experimenter can suffer from by suction-type corticosteroid, fugitive β2agonists, long-acting β2agonists, or the asthma that a combination thereof is the most appropriately controlled.Any embodiment in this article In, this experimenter can be people.

It being understood that can combine various embodiment described herein one, some, or all characteristics are to form this Other embodiment of invention.Those skilled in the art can be become apparent by these and other aspects of the invention.Pass through Detailed description below further describes these and other embodiment of the present invention.

Accompanying drawing is sketched

Fig. 1 is a sequence alignment, the comparison of display humanised antibody heavy chain's sequence.The molecular model of mice 2E2 antibody Middle-range CDRInterior Framework residues is represented by *；Away from CDRInterior residue and VCI different in receptor people's framework are residual Base highlights with ^, and these residues in receptor people's framework are become the back mutation of mice and indicated by@；Thin from the body of people's germline A ° instruction is crossed in cytoplasmic process accommodation；If those residues are different from AF471521FW and mou2E2 (i.e. mice 2E2) or with AF471521 frame Frame is different and identical with mou2E2 (i.e. mice 2E2), then by the corresponding residue back mutation human germline of receptor people's framework also Pass through+indicate.The sequence highlighted in heavy chain pattern 2 represents mou2E2 (i.e. mice 2E2) CDR and enters immediate people germline family The direct grafting of (the most immediate Germline sequences).M2E2VH corresponds to SEQ ID NO:1；People FW AF471521 corresponds to SEQ ID NO:120；Germline X92218VH366 corresponds to SEQ ID NO:121；2E2RHA corresponds to SEQ ID NO:2；2E2RHB pair Should be in SEQ ID NO:3；2E2RHC corresponds to SEQ ID NO:4；2E2RHD corresponds to SEQ ID NO:5；2E2RHE corresponds to SEQ ID NO:6；2E2RHF corresponds to SEQ ID NO:7；2E2RHG corresponds to SEQ ID NO:8；IGHV4-59FW corresponds to SEQ ID NO:122；2E2RHA2 corresponds to SEQ ID NO:9；And 2E2RHB2 is corresponding to SEQ ID NO:10.

Fig. 2 is a sequence alignment, the comparison of display humanized antibody light chain's sequence.The molecular model of mice 2E2 antibody Middle-range CDRInterior Framework residues is represented by *；Away from CDRInterior residue and VCI residues different in receptor people's framework Pass through+indicate；These residues in people FW are become the back mutation of mice and are indicated by@；Become in X93721 framework with little The back mutation of people's germline of the residue that Mus is different is indicated by #.M2E2VK corresponds to SEQ ID NO:15；X93721 corresponds to SEQ ID NO:123；Germline X01668 corresponds to SEQ ID NO:124；M2E2RKA corresponds to SEQ ID NO:16；m2E2RKB Corresponding to SEQ ID NO:17；M2E2RKC corresponds to SEQ ID NO:18；M2E2RKD corresponds to SEQ ID NO:19； M2E2RKE corresponds to SEQ ID NO:20；M2E2RKF corresponds to SEQ ID NO:21；And m2E2RKG is corresponding to SEQ ID NO: 22。

Fig. 3 is a sequence alignment, the comparison of the CDR sequence that display humanized antibody gently and suddenlys change in heavy chains.? Variant is mutated into the CDR residue closer to Germline sequences indicate with #.2E2RKA corresponds to SEQ ID NO:16；2E2RKF pair Should be in SEQ ID NO:21；2E2RKA F-Y suddenlys change corresponding to SEQ ID NO:23；2E2RKF F-Y suddenlys change corresponding to SEQ ID NO:24；2E2RHA corresponds to SEQ ID NO:2；2E2RHE corresponds to SEQ ID NO:6；2E2RHE S-G suddenlys change corresponding to SEQ ID NO:11；2E2RHE E-D suddenlys change corresponding to SEQ ID NO:12；2E2RHE Y-V suddenlys change corresponding to SEQ ID NO:13；And 2E2RHE E-D triple mutant corresponds to SEQ ID NO:14.

Fig. 4 is a width figure, is shown in institute's temp. displaying function and heats 10 minutes, is subsequently cooled to 4 DEG C, implements Siglec-8 knot afterwards Close ELISA, purification by chimeric 2C4 or 2E2RHE and 2E2RKA, 2E2RKF, 2E2RKA CDR3 mutant, or 2E2RKF The comparison that the Siglec-8 antigen of the candidate antibodies of the assembly coding of CDR3 mutant combines.

Fig. 5 is a width figure, shows the 2C4 candidate antibodies of the purification Tm compared with ch2C4 (being i.e. fitted together to 2C4 antibody).

Fig. 6 is a width figure, is shown in-20 DEG C of freezings 60 minutes and is fitted together to and the anti-Siglec-8 of humanization after room temperature is melted The stability of antibody.

Fig. 7 is a width figure, shows that anti-Siglec-8 antibody is to eosinophilic killing.At shown anti-Siglec-8 and right According in the presence of antibody concentration by total peripheral blood leucocyte incubation 16 hours.By flow cytometry monitoring eosinophil numbers Reduce, and as having high sidescattering (SSC^High) the loss of CD16 feminine gender IL5R α+cell quantify.

Fig. 8 is a series of figures, shows the apoptosis in vitro of anti-Siglec-8 antibody on human mastocyte and internal abatement.Figure 8A.HEKA IgG4 antibody, the HEKA IgG1 antibody of non-fucosylation and low fucose be fitted together to 1H10IgG1 antibody to from The antibody cell-mediated for NK of the primary people's mastocyte transplanting the peritoneal lavage of the NSGS mice having human hematopoietic stem cell depends on Rely cytotoxicity (ADCC) activity of sexual cell mediation, represented by 48 little LDH releases constantly.Comparison instruction fucosylation The human IgG1's isotype antibody not combining Siglec-8.Fig. 8 B.The abatement of internal Siglec-8 Positive Mast Cells.Use HEKA IgG4 antibody, the HEKA IgG1 antibody of non-fucosylation, Mus 1C3 antibody, or do not combine human IgG 4 isotype of Siglec-8 Control antibodies intraperitoneal process with the suitable level selective expression on mast cell surface on people's mastocyte The Siglec-8 transgenic mice of Siglec-8.N=4 mice often group.

Fig. 9 is a width figure, I type hypersensitivity reaction in the mice of display humanization anti-Siglec-8IgG4 antibody on human source Suppression.By with anti-NP-IgE (being delivered to auris dextra) or PBS control (being delivered to left ear) sensitization and execute for 24 hours after sensitization With NP-BSA, the ear transplanting NSGS mice induces passive cutaneous anaphylaxis, PCA.First 24 hours of sensitization (being indicated by *) or quick Change latter 2 hours (being indicated by@) with HEKA IgG4 or do not combine Siglec-8 human IgG 4 Isotype control antibodies process little Mus.Show latter 3 hours of attack or the Change in Mean of the 24 little thickness of ear constantly and standard error.HEKA IgG4 antibody treatment Mice, n=5 mice often group；And the mice that Isotype control antibodies processes, n=4 mice often group.

Figure 10 is a series of bar diagrams, shows mouse-anti Siglec-8 monoclonal antibody 2E2,1C3, and 1H10 is to baboon and people Eosinocyte (CD49⁺CD16^-SSC^HighCell) combination, represented by flow cytometry.Bar diagram display baboon or human blood Eosinophilic number, is that every kind of antibody is drawn, with mouse IgG 1 isotype not being combined Siglec-8 for fluorescence intensity Control antibodies compares.

Detailed Description Of The Invention

I. define

Before describing the present invention in detail, it should be understood that the invention is not restricted to particular composition or system biology, they It is of course possible to be varied from.Will also be understood that the term used herein purpose merely for description particular, not Intention is limited.As this specification and claims use, singulative " ", " a kind of " and " being somebody's turn to do " Including plural number indication thing, unless expressly stated otherwise,.So, such as, mention " a/kind molecule " optionally include two/kind or The combination of more/kind of this quasi-molecule, like this.

As used in this article, term " about " refers to that the conventional of respective value that this technical field technical staff is readily apparent that misses Difference scope." about " numerical value referred to herein or parameter include that (and description) relates to this numerical value or the embodiment of parameter itself.

Being understood by, various aspects and the embodiment of invention described herein include " comprising ", " by ... composition ", and " substantially by ... composition " aspect and embodiment.

Term " antibody " includes that polyclonal antibody, monoclonal antibody (include that the total length with immunoglobulin fc region resists Body), there is the specific antibody compositions of multi-epitope, multi-specificity antibody (such as bi-specific antibody), double antibody, and strand Molecule, and antibody fragment (such as Fab, F (ab ')₂, and Fv).Term " immunoglobulin " (Ig) in this article can with " antibody " Exchange and use.

It is different four poly-that the heavy chain (H) that 4 basic chain antibody unit are identical with two by two identical light chains (L) is constituted Body glycoprotein.IgM antibody is made up of the other polypeptide of 5 basic different tetramer unit and referred to as J chain, and comprises 10 and resist Former binding site, and IgA antibody comprises 2-5 with the combination of J chain and can be polymerized and form basic 4 chain elements of multivalence assemblage. In the case of IgG, 4 chain elements typically about 150,000 dalton.Every light chain is by a covalent disulfide bonds and heavy chain phase Even, and two heavy chains are connected with each other by one or more disulfide bond, and the number of disulfide bond depends on the isotype of heavy chain.Every Heavy chain and light chain also have disulphide bridges in the chain of regular interval.Every heavy chain has a variable domain (V at N-end_H), then It is three (for α and γ chains) or four (for μ and ε isotype) constant domain (C_H).Every light chain has one at N-end Variable domain (V_L), it is followed by a constant domain (C of its other end_L)。V_LWith V_HArranged together, and C_LWith heavy chain the first constant domain (C_H1) arranged together.Think that specific amino acid residue forms interface between light chain and heavy chain variable domain.One V_HWith one Individual V_LMatch together and form an antigen binding site.About structure and the character of different classes of antibody, see for example Basic And Clinical Immunology, the 8th edition, Daniel P.Sties, Abba I.Terr and Tristram G.Parsolw (compiles), Appleton&Lange, Norwalk, CT, page 1994,71 and the 6th chapter.

According to its constant domain aminoacid sequence, the L chain from any invertebrate species can be included into two kinds of completely different classes One in type, referred to as Kappa (κ) and lambda (λ).According to its heavy-chain constant domains (C_H) aminoacid sequence, immunoglobulin can It is included into different classifications or isotype.There are five immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, are respectively provided with referred to as α, δ, The heavy chain of ε, γ and μ.According to C_HSequence and the smaller difference of function, γ and α class can be further divided into subclass, the such as mankind and express Following subclass: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.IgG1 antibody can exist with multiple Polymorphic variant, is referred to as Allotype (summary is shown in Jefferis and Lefranc 2009.mAbs Vol 1Issue 4 1-7), they are arbitrary the suitableeest Together in using in the present invention.Common allotypic variants in crowd is exactly that those pass through letter a, and f, n, z represent.

" separate " antibody and refer to the most identified, from a kind of composition of its build environment (the most natural or restructuring) separately and/or The antibody reclaimed.In some embodiments, the polypeptide of separation does not associate with other compositions all of its build environment.It is raw The contaminative composition (being such as derived from restructuring transfectional cell) becoming environment is the research that would generally disturb antibody, diagnoses or treats use The material on way, and can include enzyme, hormone, and other oroteins character or the solute of non-proteinaceous.Implement at some In scheme, peptide purification to (1) antibody more than 95% by weight, as measured by such as Lowry method, and at some In embodiment, by weight more than 99%；(2) be enough to by using spinning cup sequenator to obtain the N end of at least 15 residues Or the degree of internal amino acid sequence；Or (3) homogenizing, use according to the SDS-PAGE under irreducibility or reductive condition and examine horse This blue or silver staining agent.The antibody separated includes the interior antibody in situ of reconstitution cell, because at least one of the natural surroundings of antibody Composition does not haves.But, the polypeptide of separation or antibody are generally prepared by least one purification step.

Term " monoclonal antibody " refers to the antibody obtained from the antibody of a group substantially homogeneity as used herein, i.e. constitutes Each antibody of colony is identical, except suddenling change and/or post translational modification (example with possible the naturally occurring of indivisible existence Such as isomerization, amidatioon) outward.In some embodiments, monoclonal antibody has C end-grain cutting at heavy chain and/or light chain and cuts.Example As, the C end-grain cutting at heavy chain and/or light chain cuts 1,2,3,4, or 5 amino acid residues.In some embodiments, C end is cut from Heavy chain removes C end lysine.In some embodiments, monoclonal antibody has N end-grain cutting at heavy chain and/or light chain and cuts.Example As, the N end-grain cutting at heavy chain and/or light chain cuts 1,2,3,4, or 5 amino acid residues.In some embodiments, clipped form Monoclonal antibody can be generated by recombinant technique.In some embodiments, monoclonal antibody is high degree of specificity, For single antigenic site.In some embodiments, monoclonal antibody is high degree of specificity, for multiple antigenicity positions Point (such as bi-specific antibody or multi-specificity antibody).The antibody population of modifier " monoclonal " instruction antibody basically homogeneity The feature obtained, should not be construed as requirement and generates antibody by any ad hoc approach.Such as, need according to present invention use Monoclonal antibody can be generated by multiple technologies, including such as hybridoma, recombinant DNA method, display technique of bacteriophage, and uses In generating in there is the animal of gene of part or whole human immunoglobulin gene's seat or encoding human immunoglobulin's sequence People or the technology of human-like antibodies.

Term " naked antibody (exposed antibody) " refers to not put together the antibody of cytotoxicity module or radioactive marker.

Term " full length antibody ", " complete antibody " and " whole antibody " is used interchangeably, and refers to the antibody of essentially completed form, Different from antibody fragment.Specifically, complete antibody includes that those have heavy chain and light chain (including Fc district).Constant domain is permissible It is native sequences constant domain (such as naive sequence constant domain) or its amino acid sequence variation.In some situation, completely resist Body can have one or more effector functions.

" antibody fragment " comprises a part for complete antibody, and the antigen of complete antibody combines and/or variable region.Antibody fragment Example include Fab, Fab ', F (ab ')₂With Fv fragment；Double antibody；Linear antibodies (see United States Patent (USP) No.5,641,870, Embodiment 2；Zapata etc., Protein Eng.8 (10): 1057-1062 (1995))；Single-chain antibody molecules；And by antibody fragment The multi-specificity antibody formed.

Produce two identical Fabs with Papain digestion of antibodies, referred to as " Fab " fragment, and one residual Remaining " Fc " fragment, its title reflects it and is prone to the ability of crystallization.Fab fragment is by complete L chain and H chain variable region territory (V_H) and weight Chain the first constant domain (C_H1) composition.It is unit price that each Fab fragment combines for antigen, and i.e. it has an antigen bound site Point.Pepsin antibody one bigger F of generation (ab ')₂Fragment, it about the same in two by disulfide bond be connected Fab fragment, has different antigen-binding activity and still is able to crosslinking antigen.Fab ' fragment is because of at C_HThe carboxyl terminal of 1 domain Add minority residue and different with Fab fragment, including the one or more cysteine from antibody hinge region. Fab '-SH is the appellation of the Fab ' that wherein constant domain cysteine residues carries free sulphur alcohol radical herein.F(ab’)₂Antibody Fragment initially generates as paired Fab ' fragment, has hinge cysteine between Fab ' fragment.Also know antibody sheet Other chemical coupling of section.

Fc fragment comprises the carboxy-terminal sections of two the H chains kept together by disulfide bond.The effector merit of antibody Sequence in Neng You Fc district determines, this region is also by Fc receptor (FcR) identification found on certain form of cell.

" Fv " is the minimum antibody fragment comprising intact antigen identification and binding site.This fragment by closely, non-covalent knot The heavy chain variable domain closed and the dimer composition of a light chain variable domain.Folding from the two domain In folded, hair growth promoting goes out six Gao Bianhuan (heavy chain and each 3 rings of light chain), contributes the amino acid residue of antigen combination and gives antibody With antigen-binding specificity.But, even single variable domain (or only comprising half Fv of to antigen-specific three HVR) is also Have and identify and the ability of conjugated antigen, although affinity is less than entire binding site.

" scFv ", is also abbreviated as " sFv " or " scFv ", is the V comprising and connecting into a polypeptide chain_HAnd V_LAntibody structure The antibody fragment in territory.In some embodiments, sFv polypeptide is at V_HWith V_LComprising peptide linker between domain further, it makes Obtain sFv and can form the desired structure of conjugated antigen.Summary about sFv sees Pluckthun, in " The Pharmacology of Monoclonal Antibodies ", volume 113, Rosenburg and Moore compiles, Springer- Verlag, New York, the 269-315 page, 1994.

" functional fragment " of antibody of the present invention comprises a part for complete antibody, generally comprises the antigen knot of complete antibody Close or the Fc district retaining or having improvement FcR binding ability of variable region or antibody.The example of antibody fragment includes linear antibodies, Single-chain antibody molecules and the multi-specificity antibody formed from antibody fragment.

Monoclonal antibody the most clearly includes " being fitted together to " antibody (immunoglobulin), wherein heavy chain and/or light chain A part is identical with derived from individually defined thing species or genus corresponding sequence in the antibody of specific antibodies classification or subclass or homology, and The remainder of chain with derived from another species or belong to another antibody isotype or subclass antibody in corresponding sequence identical or Homology, and the fragment of this antibody-like, if they show desired biologic activity (United States Patent (USP) No.4,816,567； Morrison etc., Proc.Natl.Acad.Sci.USA 81:6851-6855 (1984)).Chimeric antibody interested Including " primatized " antibody, wherein the antigen binding domain of antibody is derived from by using antigen immune stump-tailed macaque interested (macaque monkey) and the antibody that generates.As used herein, " humanized antibody " is of " chimeric antibody " Subset.

" humanization " form of inhuman (such as Mus) antibody refers to that bottom line comprises the sequence derived from non-human immunoglobulin The chimeric antibody of row.In one embodiment, the HVR residue during humanized antibody refers to human normal immunoglobulin's (receptor antibody) is used There is non-human species's (donor antibody) (such as mice, rat, rabbit or the inhuman spirit of expectation specificity, affinity and/or ability Long class animal) HVR residue replace immunoglobulin.In some situation, by the FR residue of human normal immunoglobulin with corresponding Non-human residues replace.Additionally, humanized antibody can be included in the residue not found in receptor antibody or in donor antibody. Carrying out these modifications can be the performance in order to improve antibody further, such as binding affinity.It is said that in general, humanized antibody To comprise at least one, usual two following variable domains the most whole, the most all or essentially all high change rings are corresponding In the Gao Bianhuan of non-human immunoglobulin sequence, and all or essentially all FR district is the FR district of human normal immunoglobulin's sequence, Although FR district can comprise at one or many places improves the indivedual of antibody performance (such as binding affinity, isomerization, immunogenicity etc.) FR residue substitutes.In some embodiments, the number of these amino acid replacements in FR, H chain is less than at 6, L chain is less than At 3.Humanized antibody the most also will comprise at least part of constant region for immunoglobulin (Fc), it is common that the perseverance of human normal immunoglobulin Determine district.More details see for example Jones etc., Nature 321:522-525 (1986)；Riechmann etc., Nature 332: 323-329(1988)；And Presta, Curr.Op.Struct.Biol.2:593-596 (1992).Referring also to such as Vaswani and Hamilton,Ann.Allergy,Asthma&Immunol.1:105-115(1998)；Harris, Biochem.Soc.Transactions 23:1035-1038(1995)；Hurle and Gross, Curr.Op.Biotech.5:428-433(1994)；And United States Patent (USP) No.6,982,321 and 7,087,409.Implement at some In scheme, humanized antibody is for single antigenic site.In some embodiments, humanized antibody is for multiple antigenicities Site.A kind of alternative humanization approach is recorded in United States Patent (USP) No.7,981,843 and U.S. Patent Application Publication text No.2006/0134098。

" variable region " or " variable domain " of antibody refers to the amino terminal domain of heavy chain of antibody or light chain.Heavy chain variable domain and Light-chain variable domain is properly termed as " VH " and " VL " respectively.These domains are usually the variable portion of antibody (relative to same For other antibody of class) and comprise antigen binding site.

Term " hypervariable region ", " HVR " or " HV " refer to as used herein in antibody variable domains in sequence alterable height and/or Form the region of the ring defined in structure.Generally, antibody comprise six HVR: three in VH (H1, H2, H3), three in VL (L1, L2, L3).In natural antibody, H3 and L3 shows the maximum multiformity of these six HVR, and thinks that particularly H3 is composing Give antibody so that accurate specificity to play unique effect.See for example Xu etc., Immunity 13:37-45 (2000)；Johnson And Wu, in: Methods in Molecular Biology 248:1-25 (Lo compiles, Human Press, Totowa, NJ, 2003).It is true that the camelid antibody that naturally occurs only being made up of heavy chain is to have function and stable when lacking light chain. See for example Hamers-Casterman etc., Nature 363:446-448 (1993)；And Sheriff etc., Nature Struct.Biol.3:733-736(1996)。

Used herein and contain the narration of many HVR.HVR as Kabat complementary determining region (CDR) is to become with sequence Based on the opposite sex, and it is the most frequently used (Kabat etc., Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda, MD.(1991)).Chothia HVR changes referring to position (the Chothia and Lesk J.Mol.Biol.196:901-of structure ring into 917(1987))." contacting " HVR is based on the analysis to obtainable complex crystal structure.Hereafter have recorded these The residue of each in HVR.

Except as otherwise noted, variable domain residue (HVR residue and framework residues) is according to Kabat etc., and see above numbering 's.

Those residues that " framework " or " FR " residue refers in variable domain in addition to HVR residue as defined herein.

Statement " according to the variable domain residue numbering of Kabat " or " according to the amino acid position number mode of Kabat " And variant refers to Kabat etc., for heavy chain of antibody variable domain or the numbering system of light-chain variable domain editor in seeing above.Use this Numbering system, actual linear amino acid sequence can comprise less or other aminoacid, corresponding to variable domain FR or the contracting of HVR Short or insert.Such as, the single amino acid after heavy chain variable domain can comprise H2 residue 52 inserts (being residue 52a according to Kabat) And insertion residue after heavy chain FR residue 82 (such as be residue 82a, 82b and 82c etc. according to Kabat).The Kabat of given antibody Residue numbering can be by determining antibody sequence with " standard " Kabat numbered sequence contrast homology region.

For purpose herein, " receptor people's framework " refers to comprise and has framework from human normal immunoglobulin's framework or people and derive The framework of aminoacid sequence of VL or VH framework.The receptor people's framework or the people that " derive " from human normal immunoglobulin's framework have frame Frame can comprise the aminoacid sequence that it is identical, or it can be containing the aminoacid sequence change previously existed.Real at some Executing in scheme, the number of the aminoacid change previously existed is 10 or less, 9 or less, 8 or less, 7 or less, 6 or more Few, 5 or less, 4 or less, 3 or less, or 2 or less.

It is defined as aligned sequences and in necessity about " percentage ratio (%) amino acid sequence identity " with reference to peptide sequence Time introduce breach to obtain after largest percentage sequence iden, and any conservative replacement is not considered as one of sequence iden Timesharing, the percentage rate of amino acid residue identical with reference to the amino acid residue in peptide sequence in candidate sequence.For measuring hundred The comparison of proportion by subtraction amino acid sequence identity purpose can be carried out with the various ways in the range of art technology, such as, use the public Available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Art technology Personnel can determine the suitable parameter for aligned sequences, obtains high specific to required any calculation including to institute's comparative sequences total length Method.Such as, given aminoacid sequence A relative to (to), and (with), or the % for (against) given aminoacid sequence B Amino acid sequence identity (or can be expressed as having or comprise relative to, with, or for certain of given aminoacid sequence B The given aminoacid sequence A of one % amino acid sequence identity) calculated as below:

Mark X/Y takes advantage of 100

Wherein X is to be the amino acid residue of identical match by alignment programs scoring in A and B of this program contrasts Number, and the total amino acid residues during wherein Y is B.If it will be appreciated that the length of aminoacid sequence A and the length of aminoacid sequence B Spend unequal, then the A % amino acid sequence identity relative to B will be equal to the B % amino acid sequence identity relative to A.

" in conjunction with " or " specific binding " specific polypeptide or specific polypeptide on epi-position or the antibody of its " special " is referred to In conjunction with the epi-position on this specific polypeptide or specific polypeptide and the most do not combine other polypeptide any or the antibody of polypeptide epitope.? In some embodiments, anti-Siglec-8 antibody described herein is less than this antibody pair to the combination of unrelated non-Siglec-8 polypeptide About the 10% of the combination of Siglec-8, as surveyed by means known in the art (such as enzyme-linked immunosorbent assay (ELISA)) Amount.In some embodiments, in conjunction with Siglec-8 (Siglec-8Fc fusion protein (the SEQ ID of such as dimeric forms NO:74) antibody) has≤1 μM ,≤100nM ,≤10nM ,≤2nM ,≤1nM ,≤0.7nM ,≤0.6nM ,≤0.5nM ,≤ 0.1nM ,≤0.01nM, or≤0.001nM (such as 10^-8M or less, such as 10^-8M to 10^-13M, such as 10^-9M to 10^-13M) Dissociation constant (Kd).

As used in this article, term " Siglec-8 " refers to people's Siglec-8 albumen.This term also includes Siglec-8's Natural generation variant, including splice variant or allelic variant.The aminoacid sequence of a kind of exemplary people Siglec-8 is shown in SEQ ID NO:72.The aminoacid sequence of another kind of exemplary people Siglec-8 is shown in SEQ ID NO:73.In some embodiments In, people's Siglec-8 albumen comprises the people's Siglec-8 extracellular domain being fused to immunoglobulin fc region.A kind of exemplary fusion The aminoacid sequence of people's Siglec-8 extracellular domain to immunoglobulin fc region is shown in SEQ ID NO:74.SEQ ID NO:74 In be underlined aminoacid sequence instruction Siglec-8Fc fusion protein aminoacid sequence Fc district.

People's Siglec-8 aminoacid sequence

GYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQGRFQLLGDI WSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHSRNLTCSVPW ACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPPWNLTMTVFQ GDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVHVRDEGEFTCRAQNA QGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGAGATALAFLSFCIIFIIVRSCRKKSARPAAGVGDTGMEDAKAIR GSASQGPLTESWKDGNPLKKPPPAVAPSSGEEGELHYATLSFHKVKPQDPQGQEATDSEYSEIKIHKRETAETQACL RNHNPSSKEVRG (SEQ ID NO:72)

People's Siglec-8 aminoacid sequence

GYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQGRFQLLGDI WSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHPRNLTCSVPW ACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPPWNLTMTVFQ GDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVHVRDEGEFTCRAQNA QGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGAGATALAFLSFCIIFIIVRSCRKKSARPAAGVGDTGMEDAKAIR GSASQGPLTESWKDGNPLKKPPPAVAPSSGEEGELHYATLSFHKVKPQDPQGQEATDSEYSEIKIHKRETAETQACL RNHNPSSKEVRG (SEQ ID NO:73)

Siglec-8Fc fusion protein aminoacid sequence

GYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQGRFQLLGDI WSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHSRNLTCSVPW ACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPPWNLTMTVFQ GDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVHVRDEGEFTCRAQNA QGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGIEGRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:74)

" apoptosis-induced " or " apoptotic " antibody refer to those mensuration according to standard apoptosis assays, induction of programmed The antibody of cell death, described algoscopy such as annexin V combines, DNA break, cellular contraction, and endoplasmic reticulum expands, and cell breaks Split, and/or membrane vesicle (referred to as apoptotic body) is formed.Such as, the apoptosis activity of the present invention anti-Siglec-8 antibody can be joined by film Cell dyeing is shown by albumen V.

Antibody " effector functions " refers to that those are attributable to antibody Fc district (native sequences Fc district or amino acid sequence variation Fc District) and the biologic activity that changes with antibody isotype.The example of antibody mediated effect device function includes: C1q combines and complement depends on Rely sexual cell toxicity；Fc receptor combines；The cytotoxicity (ADCC) of antibody dependent cellular mediation；Phagocytosis；Cell surface Receptor (such as B-cell receptor) is lowered；Activate with B cell.

" cytotoxicity of antibody dependent cellular mediation " or " ADCC " refer to wherein be attached to some cytotoxic cell (example Such as NK cell (NK) cell, neutrophil(e) cell and macrophage) present on secreting type Ig on Fc receptor (FcR) make this A little cytotoxic effect cells specific binding can carry the target cell of antigen, kills the thin of target cell with cytotoxin subsequently Cellular toxicity form.This antibody " equips with arms " (arm) cytotoxic cell, and is killed required by target cell by this mechanism. The main cell of mediation ADCC, NK cell, only expression Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.It is thin that Ravetch and Kinet, Annu.Rev.Immunol.9:457-92 (1991) the 464th page table 3 summarizes hemopoietic FcR on born of the same parents expresses.In some embodiments, anti-Siglec-8 antibody described herein strengthens ADCC.In order to purpose of appraisals is divided The ADCC activity of son, can carry out external ADCC algoscopy, such as United States Patent (USP) No.5, and 500,362 or 5, described in 821,337 's.The effector lymphocyte that can be used for this type of algoscopy includes PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) and NK cell (NK) cell.Or Person/it addition, can the ADCC activity of purpose of appraisals molecule in vivo, such as in animal model, such as Clynes et al., Disclosed in PNAS USA 95:652-656 (1998).Change other Fc variant bag of ADCC activity and other antibody characteristic Include Ghetie et al., Nat Biotech.15:637-40,1997；Duncan et al,Nature 332:563-564, 1988；Lund et al.,J.Immunol 147:2657-2662,1991；Lund et al,Mol Immunol 29:53- 59,1992；Alegre et al,Transplantation 57:1537-1543,1994；Hutchins et al.,Proc Natl.Acad Sci USA 92:11980-11984,1995；Jefferis et al,Immunol Lett.44:111-117, 1995；Lund et al.,FASEB J9:115-119,1995；Jefferis et al,Immunol Lett 54:101- 104,1996；Lund et al,J Immunol 157:4963-4969,1996；Armour et al.,Eur J Immunol 29:2613-2624,1999；Idusogie et al,J Immunol 164:4178-4184,200；Reddy et al,J Immunol 164:1925-1933,2000；Xu et al.,Cell Immunol 200:16-26,2000；Idusogie et al,J Immunol 166:2571-2575,2001；Shields et al.,J Biol Chem 276:6591-6604, 2001；Jefferis et al,Immunol Lett 82:57-65.2002；Presta et al.,Biochem Soc Trans 30:487-490,2002；Lazar et al.,Proc.Natl.Acad.Sci.USA 103:4005-4010,2006； United States Patent (USP) No.5,624,821；5,885,573；5,677,425；6,165,745；6,277,375；5,869,046；6,121, 022；5,624,821；5,648,260；6,194,551；6,737,056；6,821,505；6,277,375；7,335,742；With 7,317,091 those disclosed.

Term " Fc district " is in this article for defining the C petiolarea of heavy chain immunoglobulin, including native sequences Fc district and change Different Fc district.Although the border in heavy chain immunoglobulin Fc district can change, but human IgG heavy chain Fc district is normally defined from it The amino acid residue of Cys226 or Pro230 position is to the section of carboxyl terminal.It is suitable in antibody of the present invention the native sequences used Fc district includes human IgG1, IgG2, IgG3 and IgG4.(S228P, according to Kabat numbering side can to introduce single amino acid replacement Formula；It is referred to as IgG4Pro) to eliminate the heterogeneity observed in restructuring IgG4 antibody.See Angal, S.et al. (1993) Mol Immunol 30,105-108。

" non-fucosylation " or " fucose defect " antibody refers to comprise the glycosylated antibodies variant in following Fc district, its In be attached to the carbohydrate structure in Fc district and there is the fucose of minimizing or lack fucose.In some embodiments, tool The fucose being reduced or the antibody lacking fucose have the ADCC function of improvement.Identical anti-relative to what cell line generated The amount of fucose on body, non-fucosylation or the antibody of fucose defect there is the fucose of minimizing.It is contemplated herein Non-fucosylation or the antibody compositions of fucose defect be following compositions, the most less than about 50% be attached to combination The polysaccharide that in thing, the N in the Fc district of antibody connects comprises fucose.

Rock algae is there is in term " fucosylation " or " fucosylation " in referring to be attached to the oligosaccharide of the peptide main chain of antibody Saccharide residue.Specifically, the antibody of fucosylation is interior in one or both of oligosaccharide of N connection being attached to antibody Fc district The fucose that α (l, 6) connects, the position Asn in such as human IgG1 Fc territory is comprised at portion's N-acetyl glucosamine (GlcNAc) residue 297 (the EU numberings of Fc district residue).Owing to the minor sequence in immunoglobulin makes a variation, Asn297 can also be positioned at the 297 upstreams or about+3, downstream aminoacid, i.e. between the 294th and 300.

" degree of fucosylation " refers to all oligosaccharide identified with respect to means known in the art, fucosylation The percentage ratio of oligosaccharide, such as, flown by substance assistant laser desorpted-ionization in the antibody compositions that N-glycosidase F processes Time mass spectrum art (MALDI TOF MS) is assessed.In the compositions of " antibody of complete fucosylation ", essentially all Oligosaccharide comprises fucosyl residues, is i.e. fucosylation.In some embodiments, the group of the antibody of complete fucosylation Compound has the fucosylation degree of at least about 90%.Thus, the antibody in such composition is individual generally in the two of Fc district Each of the oligosaccharide that individual N connects comprises fucosyl residues.On the contrary, in the compositions of " complete non-fucosylation " antibody In, substantially none oligosaccharide is fucosylation, and what individual two N in Fc district of the antibody in such composition connected Without fucosyl residues in each of oligosaccharide.In some embodiments, the compositions of the antibody of complete non-fucosylation There is the fucosylation degree of less than about 10%.In the compositions of " antibody of part fucosylation ", only part is few Sugar comprises fucose.Both oligosaccharide that the individual N in Fc district of antibody in such composition connects, arbitrary or none in comprise rock Algae saccharide residue, premise be said composition be not included in Fc district N connect oligosaccharide in lack fucosyl residues substantially institute Having antibody individual, the N-being the most not included in Fc district connects the essentially all antibody containing fucosyl residues in both oligosaccharide Body.In one embodiment, the compositions of the antibody of part fucosylation has about 10% to about 80% (e.g., from about 50% To about 80%, about 60% to about 80%, or about 70% to about 80%) fucosylation degree.

As used in this article, " binding affinity " refers to the single binding site spouse in connection of molecule (such as antibody) The intensity of noncovalent interaction between (such as antigen).In some embodiments, to Siglec-8, (it can be two to antibody Aggressiveness, all Siglec-8-Fc fusion protein as described herein) affinity typically can pass through dissociation constant (Kd) and represent. Affinity can be measured by the common method that this area is known, including those described herein.

As used in this article, " binding affinity " refers to multiple binding sites spouse in connection of molecule (such as antibody) The bond strength of (such as antigen).

" separation " encode the nucleic acid molecules of antibody herein refer to the most identified and with generate in its environment the most therewith At least one separate nucleic acid molecules of contaminative nucleic acid molecules of association.In some embodiments, the nucleic acid of separation and syngenesis The all the components becoming environment relevant does not associate.The coding polypeptide herein separated and the nucleic acid molecules of antibody are in and are different from Form when finding it in nature or background.Therefore naturally occurring coding is the most with cell for the nucleic acid molecules separated The nucleic acid of peptide and antibody is had any different.

Term " pharmaceutical formulation " refers to that its form allows the biologic activity of active component to be effective, and without to executing The prepared product of other composition of unacceptable toxicity is produced with the experimenter of this preparaton.This type of preparaton is aseptic.

" carrier " includes pharmaceutics acceptable carrier, excipient or stabilizer as used herein, and they are being used Dosage and concentration to being exposed to its cell or mammal is nontoxic.Generally, physiology's acceptable carrier is that pH delays Bath solution.The example of physiology's acceptable carriers includes buffer agent, such as phosphate, citrate and other organic acid；Anti- Oxidant, including ascorbic acid；Low-molecular-weight (less than about 10 residues) polypeptide；Protein, such as serum albumin, gelatin or Immunoglobulin；Hydrophilic polymer, such as polyvinylpyrrolidone；Aminoacid, such as glycine, glutamine, Radix Asparagi acyl Amine, arginine or lysine；Monosaccharide, disaccharide and other carbohydrate, including glucose, mannose or dextrin；Chelating agen, all Such as EDTA；Sugar alcohol, such as mannitol or sorbitol；Become salt gegenion, such as sodium；And/or nonionic surfactant, such as TWEEN^TM, Polyethylene Glycol (PEG) and PLURONICS^TM。

As used in this article, term " is treated " or " process " refers to be designed in the process of clinical pathology changing institute The clinical intervention of the nature process for the treatment of individuality or cell.The desired effects for the treatment of includes the speed slowing down progression of disease, improves Or the state that palliates a disease, and exempt or improve prognosis.If one or more are with the disease (disease of such as Eosinophils mediate Sick) relevant symptom is mitigated or eliminates, then say that such as individuality is succeeded " treatment ".Such as, if treatment causes suffering from The individual quality of life suffered from the disease improves, the decrease in dose of other medication that treatment disease needs, and reduces the frequency of palindromia Rate, the order of severity palliated a disease, postpone disease and occur or progress, and/or extend individual survival, then individuality is succeeded " treat ".

As used in this article, " with ... associating " refer to outside one treats form, use another kind for the treatment of form.As This, and " with .... associating " refer to that individuality is used one treats form before, during or after use another kind for the treatment of form.

As used in this article, term " prevents " to include to provide for the generation of disease in individuality or recurrence to take precautions against.Individual Body can tend to disease, susceptible to disease, or risky generation disease, but N-Y-D- has disease.Some embodiment party In case, use anti-Siglec-8 antibody described herein to postpone the generation of disease.

As used in this article, the individuality of " risky " generation disease can be with and without detectable disease or disease disease Shape, and can show before Therapeutic Method described herein or not show detectable disease or disease symptoms." risky " table Showing that individuality has one or more risk factor, they are the disease with Eosinophils mediate and/or mast cell mediated Disease there is relevant measurable parameter, as known in the art.The individuality with these risk factor one or more has Probability generation disease more higher than the individuality not having these risk factor one or more.

" effective dose " refer at least required dosage and effectively realize on the time effect that is desired or that specify (include treatment Or prevention result) amount.Effective dose can provide in one or many is used." therapeutically effective amount " refers at least to realize specific disease Cmin required for the measured improvement of disease.Therapeutically effective amount herein can be according to the morbid state of such as patient, year Age, sex and body weight and this antibody cause the factors such as the ability of expectation response to change in individuality.Therapeutically effective amount is all right Refer to that the treatment beneficial effect of this antibody surpasses any poisonous or amount of detrimental consequences." prevention effective dose " refer at required dosage and The amount of desired preventive effect is effectively realized on time.Typically but not necessarily, due to preventive dose be before seizure of disease or For experimenter disease early stage, therefore prevention effective dose can be less than therapeutically effective amount.

It is contrary with short term patterns that " for a long time " uses finger, uses medicine in a continuous mode, thus initial therapy effect (is lived Property) maintain longer period of time." intermittently " using finger is not the treatment being carried out continuously free of discontinuities, but the most periodic.

As used in this article, " individual " or " experimenter " is mammal.For therapeutic purposes, " mammal " includes People, domestic animal and domestic animal, and zoo, motion or pet animals, such as dog, horse, rabbit, cattle, pig, hamster, gerbil jird, mice, snow Ermine, rat, cat etc..In some embodiments, individual or experimenter refers to people.

The most anti-Siglec-8 antibody and compositions

On the one hand, the present invention provides the antibody of combination people Siglec-8 of separation.In some embodiments, described herein Anti-Siglec-8 antibody has one or more following characteristics: (1) combines people Siglec-8；(2) born of the same parents of people Siglec-8 are combined Foreign lands；(3) people Siglec-8 is combined with the affinity higher than mouse antibodies 2E2 and/or mouse antibodies 2C4；(4) with than mice Antibody 2E2 and/or mouse antibodies 2C4 wants high affinity to combine people Siglec-8；(5) in hot transfer assay method, have about 70 DEG C-72 DEG C or higher Tm；(6) there is the fucosylation of reduction degree or non-fucosylation；(7) it is combined in addicted to daybreak On erythrocyte express people Siglec-8 and induce eosinocyte apoptosis；(8) people expressed it is combined on mastocyte Siglec-8 also cuts down mastocyte；(9) it is combined on mastocyte the people Siglec-8 expressed and suppresses the Fc ε of mastocyte RI dependency activity (such as histamine release, PGD₂Release, Ca²⁺Stream, and/or β-hexosaminidase release, etc.)；(10) engineering Change transformation to improve ADCC activity；(11) it is combined on mastocyte the people Siglec-8 expressed and kills fertilizer by ADCC activity Maxicell (in vitro and/or in vivo)；(12) Siglec-8 of people and non-human primates is combined；(13) people Siglec-8 is combined Domain 1, domain 2, and/or domain 3, or combine comprise people's Siglec-8 domain 1, domain 2, and/or structure The Siglec-8 polypeptide (fusion protein the most described herein) in territory 3；(14) with the EC less than mouse antibodies 2E2 or 2C4₅₀'s EC₅₀The eosinocyte that abatement has activated.

On the one hand, the present invention provides the antibody combining people Siglec-8.In some embodiments, this people's Siglec-8 bag Aminoacid sequence containing SEQ ID NO:72.In some embodiments, this people Siglec-8 comprises the ammonia of SEQ ID NO:73 Base acid sequence.In some embodiments, the epi-position in antibodies people's Siglec-8 domain 1 described herein, wherein structure Territory 1 comprises the aminoacid sequence of SEQ ID NO:112.In some embodiments, antibodies people Siglec-8 described herein Epi-position in domain 2, wherein domain 2 comprises the aminoacid sequence of SEQ ID NO:113.In some embodiments, originally Epi-position in the described antibodies people's Siglec-8 domain 3 of literary composition, wherein domain 3 comprises the aminoacid of SEQ ID NO:114 Sequence.In some embodiments, antibodies described herein comprise SEQ ID NO:116 amino acid whose fusion protein but not In conjunction with the amino acid whose fusion protein comprising SEQ ID NO:115.In some embodiments, antibodies described herein comprises The amino acid whose fusion protein of SEQ ID NO:117 but do not combine the amino acid whose fusion protein comprising SEQ ID NO:115.? In some embodiments, amino acid whose fusion protein that antibodies described herein comprises SEQ ID NO:117 but do not combine bag Amino acid whose fusion protein containing SEQ ID NO:116.In some embodiments, antibodies people Siglec-8 described herein Linear epitope in extracellular domain.In some embodiments, the conformation in antibodies people's Siglec-8 extracellular domain described herein Epi-position.In some embodiments, antibodies described herein is expressed on eosinocyte people Siglec-8 and induce addicted to Eosinophile apoptosis.In some embodiments, the people Siglec-8 that antibodies described herein is expressed on mastocyte is also Abatement mastocyte.In some embodiments, the people Siglec-8 that antibodies described herein is expressed on mastocyte is also The activity of suppression mast cell mediated.In some embodiments, the people that antibodies described herein is expressed on mastocyte Siglec-8 also kills mastocyte by ADCC activity.In some embodiments, antibody described herein abatement mastocyte And suppress Mast cell activation.In some embodiments, antibody herein is cut down the eosinocyte activated and suppresses Mast cell activation.

The anti-Siglec-8 of the combination people Siglec-8 and non-human primates Siglec-8 of a kind of separation provided herein resists Body.There is the identifying for survey before clinical in non-human primates of anti-Siglec-8 antibody of antibody of primates cross reactivity Examination can be useful.On the one hand, the present invention provides the antibody combining non-human primates Siglec-8.On the one hand, the present invention provides In conjunction with people Siglec-8 and the antibody of non-human primates Siglec-8.In some embodiments, this non-human primates Siglec- 8 aminoacid sequence comprising SEQ ID NO:118 or its parts.In some embodiments, this non-human primates Siglec-8 Comprise aminoacid sequence or its part of SEQ ID NO:119.In some embodiments, this non-human primates is baboon (example Such as green baboon (Papio Anubis)).In some embodiments, in conjunction with people Siglec-8's and non-human primates Siglec-8 Epi-position in antibodies people's Siglec-8 domain 1.In a further embodiment, the domain of people Siglec-8 1 aminoacid sequence comprising SEQ ID NO:112.In some embodiments, in conjunction with people Siglec-8 and non-human primates Epi-position in antibodies people's Siglec-8 domain 3 of Siglec-8.In a further embodiment, people The domain 3 of Siglec-8 comprises the aminoacid sequence of SEQ ID NO:114.In some embodiments, in conjunction with people Siglec- The antibody of 8 and non-human primates Siglec-8 is humanized antibody, chimeric antibody, or people's antibody.In some embodiments, knot The antibody closing people Siglec-8 and non-human primates Siglec-8 is murine antibody.In some embodiments, in conjunction with people Siglec- The antibody of 8 and non-human primates Siglec-8 is human IgG1's antibody.

On the one hand, anti-Siglec-8 antibody described herein is monoclonal antibody.On the one hand, anti-Siglec-8 described herein resists Body is antibody fragment (including Fab), such as Fab, Fab '-SH, Fv, scFv, or (Fab ')₂Fragment.On the one hand, Anti-Siglec-8 antibody described herein is chimeric, humanization, or people's antibody.On the one hand, any anti-Siglec-8 described herein resists Body is purification.

On the one hand, it is provided that be combined the anti-Siglec-8 antibody of Siglec-8 with Mus 2E2 antibody and Mus 2C4 antibody competition.Also There is provided and Mus 2E2 antibody and the anti-Siglec-8 antibody of the identical epi-position of Mus 2C4 antibodies.On the one hand, it is provided that with described herein The anti-Siglec-8 of any anti-Siglec-8 antibody (such as HEKA, HEKF, 1C3,1H10,4F11) competition binding Siglec-8 resists Body.Also provide for combining identical table with any anti-Siglec-8 antibody (such as HEKA, HEKF, 1C3,1H10,4F11) described herein The anti-Siglec-8 antibody of position.

In one aspect of the invention, it is provided that encode the polynucleotide of anti-Siglec-8 antibody.In certain embodiments, The carrier comprising the polynucleotide encoding anti-Siglec-8 antibody is provided.In certain embodiments, it is provided that comprise examples of such carriers Host cell.In another aspect of the present invention, it is provided that comprise anti-Siglec-8 antibody or encode anti-Siglec-8 antibody The compositions of polynucleotide.In certain embodiments, the compositions of the present invention is the disease for treating Eosinophils mediate The pharmaceutical formulation of the disease (those enumerated the most herein) of disease and/or mast cell mediated.

On the one hand, the 1 of murine antibody 2C4 that comprises provided herein, 2,3,4,5, or the anti-Siglec-8 of 6 kinds of HVR sequences is anti- Body.On the one hand, the 1 of murine antibody 2E2 that comprises provided herein, 2,3,4,5, or the anti-Siglec-8 antibody of 6 kinds of HVR sequences.? In some embodiments, this HVR is Kabat CDR or Chothia CDR.

On the one hand, the 1 of murine antibody 1C3 that comprises provided herein, 2,3,4,5, or the anti-Siglec-8 of 6 kinds of HVR sequences is anti- Body.On the one hand, the 1 of murine antibody 4F11 that comprises provided herein, 2,3,4,5, or the anti-Siglec-8 antibody of 6 kinds of HVR sequences.One Aspect, the 1 of murine antibody 1H10 that comprises provided herein, 2,3,4,5, or the anti-Siglec-8 antibody of 6 kinds of HVR sequences.At some In embodiment, this HVR is Kabat CDR or Chothia CDR.

In some embodiments, the epi-position in antibodies people's Siglec-8 domain 1 described herein, wherein domain 1 aminoacid sequence comprising SEQ ID NO:112.In some embodiments, antibodies people Siglec-8 described herein knot Epi-position in structure territory 2, wherein domain 2 comprises the aminoacid sequence of SEQ ID NO:113.In some embodiments, herein Epi-position in described antibodies people's Siglec-8 domain 3, wherein domain 3 comprises the aminoacid sequence of SEQ ID NO:114 Row.

In some embodiments, antibodies described herein comprises the amino acid whose fusion protein of SEQ ID NO:116 But do not combine the amino acid whose fusion protein comprising SEQ ID NO:115.In some embodiments, antibodies described herein The amino acid whose fusion protein that comprises SEQ ID NO:117 but do not combine the amino acid whose fusion egg comprising SEQ ID NO:115 In vain.In some embodiments, antibodies described herein comprises SEQ ID NO:117 amino acid whose fusion protein but do not tie Close the amino acid whose fusion protein comprising SEQ ID NO:116.

On the one hand, provided herein is a kind of anti-Siglec-8 antibody comprising variable region of heavy chain and variable region of light chain, its In this variable region of heavy chain comprise the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, (ii) comprises SEQ ID NO: The HVR-H2 of the aminoacid sequence of 62, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:63；And/or wherein This variable region of light chain comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, and (ii) comprises SEQ ID NO:65 The HVR-L2 of aminoacid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:66.

On the one hand, a kind of anti-Siglec-8 antibody comprising variable region of heavy chain and variable region of light chain provided herein, wherein This variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises SEQ ID NO:62 The HVR-H2 of aminoacid sequence, and (iii) comprise the HVR-H3 of the aminoacid sequence selected from SEQ ID NO:67-70；And/or Wherein this variable region of light chain comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, and (ii) comprises SEQ ID The HVR-L2 of the aminoacid sequence of NO:65, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:66.

On the one hand, a kind of anti-Siglec-8 antibody comprising variable region of heavy chain and variable region of light chain provided herein, wherein This variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises SEQ ID NO:62 The HVR-H2 of aminoacid sequence, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:63；And/or wherein should Variable region of light chain comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, and (ii) comprises SEQ ID NO:65's The HVR-L2 of aminoacid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:71.

On the other hand, a kind of anti-Siglec-8 antibody comprising variable region of heavy chain and variable region of light chain provided herein, its In this variable region of heavy chain comprise the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, (ii) comprises SEQ ID NO: The HVR-H2 of the aminoacid sequence of 62, and (iii) comprise the HVR-H3 of the aminoacid sequence selected from SEQ ID NO:67-70；With/ Or wherein this variable region of light chain comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, (ii) comprises SEQ ID The HVR-L2 of the aminoacid sequence of NO:65, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:71.

On the other hand, a kind of anti-Siglec-8 antibody comprising variable region of heavy chain and variable region of light chain provided herein, its In this variable region of heavy chain comprise the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:88, (ii) comprises SEQ ID NO: The HVR-H2 of the aminoacid sequence of 91, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:94；And/or light chain Variable region, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:97, and (ii) comprises SEQ ID NO:100's The HVR-L2 of aminoacid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:103.Some embodiment party In case, the epi-position in antibodies people's Siglec-8 domain 2 described herein, wherein domain 2 comprises SEQ ID NO:113's Aminoacid sequence.

On the other hand, a kind of anti-Siglec-8 antibody comprising variable region of heavy chain and variable region of light chain provided herein, its In this variable region of heavy chain comprise the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:89, (ii) comprises SEQ ID NO: The HVR-H2 of the aminoacid sequence of 92, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:95；And/or this is light Chain variable region comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:98, and (ii) comprises the ammonia of SEQ ID NO:101 The HVR-L2 of base acid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:104.In some embodiments In, the epi-position in antibodies people's Siglec-8 domain 3 described herein, wherein domain 3 comprises the ammonia of SEQ ID NO:114 Base acid sequence.In some embodiments, antibodies people Siglec-8 and non-human primates Siglec-8 described herein.

On the other hand, a kind of anti-Siglec-8 antibody comprising variable region of heavy chain and variable region of light chain provided herein, its In this variable region of heavy chain comprise the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:90, (ii) comprises SEQ ID NO: The HVR-H2 of the aminoacid sequence of 93, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:96；And/or this is light Chain variable region comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:99, and (ii) comprises the ammonia of SEQ ID NO:102 The HVR-L2 of base acid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:105.In some embodiments In, the epi-position in antibodies people's Siglec-8 domain 1 described herein, wherein domain 1 comprises the ammonia of SEQ ID NO:112 Base acid sequence.In some embodiments, antibodies people Siglec-8 and non-human primates Siglec-8 described herein.

Anti-Siglec-8 antibody described herein can comprise any suitable framework variable domain sequence, and premise is that this resists Body retains the ability combining people Siglec-8.As used in this article, heavy chain framework regions is referred to as " HC-FR1-FR4 ", and light chain frame Frame district is referred to as " LC-FR1-FR4 ".In some embodiments, this anti-Siglec-8 antibody comprises SEQ ID NO:26, and 34,38, Heavy-chain variable domains Frame sequence with 45 (being HC-FR1, HC-FR2, HC-FR3, and HC-FR4 respectively).Implement at some In scheme, this anti-Siglec-8 antibody comprises SEQ ID NO:48, and 51,55, and 60 (it is LC-FR1 respectively, LC-FR2, LC- FR3, and LC-FR4) light variable domains Frame sequence.In some embodiments, this anti-Siglec-8 antibody comprises SEQ ID NO:48,51,58, and the light variable domains of 60 (they being LC-FR1, LC-FR2, LC-FR3, and LC-FR4 respectively) Frame sequence.

In one embodiment, anti-Siglec-8 antibody comprises the weight chain variable structure of Frame sequence and hypervariable region Territory, wherein this Frame sequence comprises is SEQ ID NO:26-29 (HC-FR1) respectively, SEQ ID NO:31-36 (HC-FR2), SEQ ID NO:38-43 (HC-FR3), and the HC-FR1-HC-FR4 sequence of SEQ ID NO:45 or 46 (HC-FR4)；HVR-H1 Comprise the aminoacid sequence of SEQ ID NO:61；HVR-H2 comprises the aminoacid sequence of SEQ ID NO:62；And HVR-H3 comprises The aminoacid sequence of SEQ ID NO:63.In one embodiment, anti-Siglec-8 antibody comprises Frame sequence and height Becoming the heavy-chain variable domains in district, wherein this Frame sequence comprises is SEQ ID NO:26-29 (HC-FR1) respectively, SEQ ID NO:31-36 (HC-FR2), SEQ ID NO:38-43 (HC-FR3), and the HC-FR1-of SEQ ID NO:45 or 46 (HC-FR4) HC-FR4 sequence；HVR-H1 comprises the aminoacid sequence of SEQ ID NO:61；HVR-H2 comprises the aminoacid of SEQ ID NO:62 Sequence；And HVR-H3 comprises the aminoacid sequence selected from SEQ ID NO:67-70.In one embodiment, anti-Siglec-8 Antibody comprises the light variable domains of Frame sequence and hypervariable region, and wherein this Frame sequence comprises is SEQ ID respectively NO:48 or 49 (LC-FR1), SEQ ID NO:51-53 (LC-FR2), SEQ ID NO:55-58 (LC-FR3), and SEQ ID The LC-FR1-LC-FR4 sequence of NO:60 (LC-FR4)；HVR-L1 comprises the aminoacid sequence of SEQ ID NO:64；HVR-L2 bag Aminoacid sequence containing SEQ ID NO:65；And HVR-L3 comprises the aminoacid sequence of SEQ ID NO:66.An embodiment party In case, anti-Siglec-8 antibody comprises the light variable domains of Frame sequence and hypervariable region, wherein this Frame sequence bag Containing is SEQ ID NO:48 or 49 (LC-FR1) respectively, SEQ ID NO:51-53 (LC-FR2), SEQ ID NO:55-58 (LC- And the LC-FR1-LC-FR4 sequence of SEQ ID NO:60 (LC-FR4) FR3),；HVR-L1 comprises the aminoacid of SEQ ID NO:64 Sequence；HVR-L2 comprises the aminoacid sequence of SEQ ID NO:65；And HVR-L3 comprises the aminoacid sequence of SEQ ID NO:71. In an embodiment of these antibody, this heavy chain variable domain comprise selected from SEQ ID NO:2-10 aminoacid sequence and should Light-chain variable domain comprises the aminoacid sequence selected from SEQ ID NO:16-22.In an embodiment of these antibody, this is heavy Aminoacid sequence and this light-chain variable domain that chain variable domain comprises selected from SEQ ID NO:2-10 comprise selected from SEQ ID NO:23 Or the aminoacid sequence of 24.In an embodiment of these antibody, this heavy chain variable domain comprises selected from SEQ ID NO:11- The aminoacid sequence of 14 and this light-chain variable domain comprise the aminoacid sequence selected from SEQ ID NO:16-22.At these antibody In one embodiment, this heavy chain variable domain comprises the aminoacid sequence selected from SEQ ID NO:11-14 and this light-chain variable domain Comprise the aminoacid sequence selected from SEQ ID NO:23 or 24.In an embodiment of these antibody, this heavy chain variable domain The aminoacid sequence comprising SEQ ID NO:6 and this light-chain variable domain comprise the aminoacid sequence of SEQ ID NO:16.At these In one embodiment of antibody, this heavy chain variable domain comprises aminoacid sequence and this light-chain variable domain bag of SEQ ID NO:6 Aminoacid sequence containing SEQ ID NO:21.

In some embodiments, heavy chain HVR sequence comprises following:

a)HVR-H1(IYGAH(SEQ ID NO:61))；

b)HVR-H2(VIWAGGSTNYNSALMS(SEQ ID NO:62))；With

c)HVR-H3(DGSSPYYYSMEY(SEQ ID NO:63)；DGSSPYYYGMEY(SEQ ID NO:67)； DGSSPYYYSMDY(SEQ ID NO:68)；DGSSPYYYSMEV(SEQ ID NO:69)；Or GSSPYYYGMDV (SEQ ID NO:70))。

In some embodiments, heavy chain HVR sequence comprises following:

a)HVR-H1(SYAMS(SEQ ID NO:88)；DYYMY(SEQ ID NO:89)；Or SSWMN (SEQ ID NO: 90))；

b)HVR-H2(IISSGGSYTYYSDSVKG(SEQ ID NO:91)；RIAPEDGDTEYAPKFQG(SEQ ID NO: 92)；Or QIYPGDDYTNYNGKFKG (SEQ ID NO:93))；With

c)HVR-H3(HETAQAAWFAY(SEQ ID NO:94)；EGNYYGSSILDY(SEQ ID NO:95)；Or LGPYGPFAD(SEQ ID NO:96))。

In some embodiments, heavy chain FR sequence comprises following:

a)HC-FR1(EVQLVESGGGLVQPGGSLRLSCAASGFSLT(SEQ ID NO:26)； EVQLVESGGGLVQPGGSLRLSCAVSGFSLT(SEQ ID NO:27)；QVQLQESGPGLVKPSETLSLTCTVSGGSIS (SEQ ID NO:28)；Or QVQLQESGPGLVKPSETLSLTCTVSGFSLT (SEQ ID NO:29))；

b)HC-FR2(WVRQAPGKGLEWVS(SEQ ID NO:31)；WVRQAPGKGLEWLG(SEQ ID NO:32)； WVRQAPGKGLEWLS(SEQ ID NO:33)；WVRQAPGKGLEWVG(SEQ ID NO:34)；WIRQPPGKGLEWIG(SEQ ID NO:35)；Or WVRQPPGKGLEWLG (SEQ ID NO:36))；

c)HC-FR3(RFTISKDNSKNTVYLQMNSLRAEDTAVYYCAR(SEQ ID NO:38)； RLSISKDNSKNTVYLQMNSLRAEDTAVYYCAR(SEQ ID NO:39)； RLTISKDNSKNTVYLQMNSLRAEDTAVYYCAR(SEQ ID NO:40)； RFSISKDNSKNTVYLQMNSLRAEDTAVYYCAR(SEQ ID NO:41)； RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO:42)；Or RLSISKDNSKNQVSLKLSSVTAADTAVYYCAR(SEQ ID NO:43))；With

d)HC-FR4(WGQGTTVTVSS(SEQ ID NO:45)；Or WGQGTLVTVSS (SEQ ID NO:46)).

In some embodiments, light chain HVR sequence comprises following:

a)HVR-L1(SATSSVSYMH(SEQ ID NO:64))；

b)HVR-L2(STSNLAS(SEQ ID NO:65))；With

c)HVR-L3(QQRSSYPFT(SEQ ID NO:66)；Or QQRSSYPYT (SEQ ID NO:71)).

In some embodiments, light chain HVR sequence comprises following:

a)HVR-L1(SASSSVSYMH(SEQ ID NO:97)；RASQDITNYLN(SEQ ID NO:98)；Or SASSSVSYMY(SEQ ID NO:99))；

b)HVR-L2(DTSKLAY(SEQ ID NO:100)；FTSRLHS(SEQ ID NO:101)；Or DTSSLAS (SEQ ID NO:102))；With

c)HVR-L3(QQWSSNPPT(SEQ ID NO:103)；QQGNTLPWT(SEQ ID NO:104)；Or QQWNSDPYT(SEQ ID NO:105))。

In some embodiments, light chain FR sequences comprises following:

a)LC-FR1(EIVLTQSPATLSLSPGERATLSC(SEQ ID NO:48)；Or EIILTQSPATLSLSPGERATLSC(SEQ ID NO:49))；

b)LC-FR2(WFQQKPGQAPRLLIY(SEQ ID NO:51)；WFQQKPGQAPRLWIY(SEQ ID NO:52)； Or WYQQKPGQAPRLLIY (SEQ ID NO:53))；

c)LC-FR3(GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC(SEQ ID NO:55)； GVPARFSGSGSGTDYTLTISSLEPEDFAVYYC(SEQ ID NO:56)； GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC(SEQ ID NO:57)；Or GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC(SEQ ID NO:58))；With

d)LC-FR4(FGPGTKLDIK(SEQ ID NO:60))。

In some embodiments, the anti-Siglec-8 antibody of a kind of specific binding people Siglec-8 provided herein (the anti-Siglec-8 of such as humanization) antibody, wherein this antibody comprises variable region of heavy chain and variable region of light chain, wherein this antibody bag Contain:

(a) heavy-chain variable domains, it comprises:

(1) HC-FR1 of aminoacid sequence selected from SEQ ID NO:26-29 is comprised；

(2) HVR-H1 of the aminoacid sequence of SEQ ID NO:61 is comprised；

(3) HC-FR2 of aminoacid sequence selected from SEQ ID NO:31-36 is comprised；

(4) HVR-H2 of the aminoacid sequence of SEQ ID NO:62 is comprised；

(5) HC-FR3 of aminoacid sequence selected from SEQ ID NO:38-43 is comprised；

(6) HVR-H3 of the aminoacid sequence of SEQ ID NO:63 is comprised；With

(7) HC-FR4 of aminoacid sequence selected from SEQ ID NO:45-46 is comprised, and/or

(b) light variable domains, it comprises:

(1) LC-FR1 of aminoacid sequence selected from SEQ ID NO:48-49 is comprised；

(2) HVR-L1 of the aminoacid sequence of SEQ ID NO:64 is comprised；

(3) LC-FR2 of aminoacid sequence selected from SEQ ID NO:51-53 is comprised；

(4) HVR-L2 of the aminoacid sequence of SEQ ID NO:65 is comprised；

(5) LC-FR3 of aminoacid sequence selected from SEQ ID NO:55-58 is comprised；

(6) HVR-L3 of the aminoacid sequence of SEQ ID NO:66 is comprised；With

(7) LC-FR4 of the aminoacid sequence of SEQ ID NO:60 is comprised.

On the one hand, provided herein a kind of comprise selected from the heavy-chain variable domains of SEQ ID NO:2-10 and/or comprise Anti-Siglec-8 antibody selected from the light variable domains of SEQ ID NO:16-22.On the one hand, one provided herein comprises Selected from the heavy-chain variable domains of SEQ ID NO:2-10 and/or comprise the light chain variable domain selected from SEQ ID NO:23 or 24 The anti-Siglec-8 antibody in territory.On the one hand, a kind of weight chain variable structure comprised selected from SEQ ID NO:11-14 provided herein Territory and/or comprise the anti-Siglec-8 antibody of the light variable domains selected from SEQ ID NO:16-22.On the one hand, herein There is provided a kind of comprise selected from the heavy-chain variable domains of SEQ ID NO:11-14 and/or comprise selected from SEQ ID NO:23 or 24 The anti-Siglec-8 antibody of light variable domains.On the one hand, a kind of heavy chain comprising SEQ ID NO:6 provided herein can Structure changes territory and/or comprise the anti-Siglec-8 antibody of the light variable domains selected from SEQ ID NO:16 or 21.

On the one hand, a kind of heavy-chain variable domains comprised selected from SEQ ID NO:106-108 provided herein and/or bag Anti-Siglec-8 antibody containing the light variable domains selected from SEQ ID NO:109-111.On the one hand, one provided herein The heavy-chain variable domains comprising SEQ ID NO:106 and/or the light variable domains comprising SEQ ID NO:109 anti- Siglec-8 antibody.On the one hand, the heavy-chain variable domains of a kind of SEQ of comprising ID NO:107 provided herein and/or comprise The anti-Siglec-8 antibody of the light variable domains of SEQ ID NO:110.On the one hand, one provided herein comprises SEQ ID The heavy-chain variable domains of NO:108 and/or comprise the anti-Siglec-8 antibody of light variable domains of SEQ ID NO:111.

In some embodiments, one provided herein anti-Siglec-8 antibody, it comprises and selected from SEQ ID The aminoacid sequence of NO:2-14 has at least 90%, and 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or The heavy-chain variable domains of the aminoacid sequence of 99% sequence iden.In some embodiments, one provided herein resists Siglec-8 antibody, it comprises and has at least 90% with selected from the aminoacid sequence of SEQ ID NO:106-108, and 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or the weight chain variable structure of the aminoacid sequence of 99% sequence iden Territory.In some embodiments, have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or The aminoacid sequence of 99% sequence iden contains replacement relative to canonical sequence, inserts, or deletes, but comprises this aminoacid The antibody of sequence retains the ability combining people Siglec-8.In some embodiments, this replacement, insert, or delete (such as 1, 2,3,4, or 5 aminoacid) in region beyond HVR (i.e. in FR) occur.In some embodiments, anti-Siglec-8 Antibody comprises the heavy-chain variable domains of the aminoacid sequence of SEQ ID NO:6.In some embodiments, anti- Siglec-8 antibody comprises the heavy-chain variable domains of the aminoacid sequence selected from SEQ ID NO:106-108.

In some embodiments, one provided herein anti-Siglec-8 antibody, it comprises and selected from SEQ ID The aminoacid sequence of NO:16-24 has at least 90%, and 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or The light variable domains of the aminoacid sequence of 99% sequence iden.In some embodiments, one provided herein resists Siglec-8 antibody, it comprises and has at least 90% with selected from the aminoacid sequence of SEQ ID NO:109-111, and 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or the light chain variable domain of the aminoacid sequence of 99% sequence iden Territory.In some embodiments, have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or The aminoacid sequence of 99% sequence iden contains replacement relative to canonical sequence, inserts, or deletes, but comprises this aminoacid The antibody of sequence retains the ability combining people Siglec-8.In some embodiments, this replacement, insert, or delete (such as 1, 2,3,4, or 5 aminoacid) in region beyond HVR (i.e. in FR) occur.In some embodiments, anti-Siglec-8 Antibody comprises the light variable domains of the aminoacid sequence of SEQ ID NO:16 or 21.In some embodiments, anti- Siglec-8 antibody comprises the heavy-chain variable domains of the aminoacid sequence selected from SEQ ID NO:109-111.

In some embodiments, one provided herein anti-Siglec-8 antibody, it comprises weight shown in Fig. 1 or Fig. 3 Chain variable domains.

In some embodiments, one provided herein anti-Siglec-8 antibody, it comprises shown in Fig. 2 or Fig. 3 light Chain variable domains.

On the one hand, the present invention provides a kind of anti-Siglec-8 antibody, and it is a kind of that it comprises (a), two kinds, or three kinds are selected from Fig. 1 Or the VH HVR of VH HVR shown in Fig. 3 and/or (b) are a kind of, two kinds, or three kinds of VL selected from VL HVR shown in Fig. 2 or Fig. 3 HVR.On the one hand, the present invention provides a kind of anti-Siglec-8 antibody, and it comprises selected from weight chain variable structure shown in Fig. 1 or Fig. 3 The heavy-chain variable domains in territory and the light variable domains selected from light variable domains shown in Fig. 2 or Fig. 3.

In some embodiments, one provided herein anti-Siglec-8 antibody, it comprises antibody (example shown in table 3 Such as HAKA antibody, HAKB antibody, HAKC antibody, etc.) heavy chain variable domain and/or light variable domains.

There are five big immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, there is the weight being called α, δ, ε, γ and μ Chain.γ and α class is further separated into subclass, and such as, people expresses following subclass: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. IgG1 antibody can exist with multiple Polymorphic variant, and referred to as (summary is shown in Jefferis and Lefranc to allotype 2009.mAbs Vol 1Issue 4 1-7), they arbitrary some embodiments being adapted in this article use.Crowd In common allotypic variants be exactly those by letter a, f, n, z or a combination thereof represent.Any enforcement in this article In scheme, this antibody can comprise the heavy chain Fc district in human IgG Fc district.In further embodiment, this human IgG Fc district Comprise human IgG1 or IgG4.In some embodiments, this human IgG 4 comprises amino acid replacement S228P, wherein amino acid residue It is according to the EU index number in Kabat.In some embodiments, this human IgG1 comprises the aminoacid of SEQ ID NO:78 Sequence.In some embodiments, this human IgG 4 comprises the aminoacid sequence of SEQ ID NO:79.

In some embodiments, one provided herein anti-Siglec-8 antibody, it comprises SEQ ID NO:75 The heavy chain of aminoacid sequence；And/or comprise the light chain of aminoacid sequence selected from SEQ ID NO:76 or 77.Implement at some In scheme, this antibody can comprise the heavy chain of the aminoacid sequence of SEQ ID NO:87；And/or comprise SEQ ID NO:76 The light chain of aminoacid sequence.In some embodiments, this anti-Siglec-8 antibody abatement mastocyte and suppression are loose thin Born of the same parents activate.In some embodiments, this anti-Siglec-8 antibody cuts down the eosinocyte activated and suppression mastocyte Activation.

1. affinity of antibody

In some respects, anti-Siglec-8 antibody described herein is with compared with mouse antibodies 2E2 and/or mouse antibodies 2C4 Roughly the same or higher affinity and/or higher affinity combine people Siglec-8.In certain embodiments, herein The anti-Siglec-8 antibody provided has≤1 μM ,≤150nM ,≤100nM ,≤50nM ,≤10nM ,≤1nM ,≤0.1nM ,≤ 0.01nM, or≤0.001nM (such as 10^-8M or less, such as 10^-8M to 10^-13M, such as 10^-9M to 10^-13M) dissociation constant (Kd).In some embodiments, anti-Siglec-8 antibody described herein is with higher than mouse antibodies 2E2 and/or mouse antibodies 2C4 About 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, the affinity of about 9 times or about 10 times combines people Siglec-8.In some embodiments in this article, this anti-Siglec-8 antibody comprises the aminoacid of SEQ ID NO:6 The variable region of heavy chain of sequence；And/or comprise the variable region of light chain of aminoacid sequence selected from SEQ ID NO:16 or 21.

In one embodiment, the binding affinity of anti-Siglec-8 antibody can be surveyed by surface plasmon resonance The method of determining measures.Such as, BIAcore can be used in 25 DEG C^TM-2000 or BIAcore^TM-3000(BIAcore,Inc., Piscataway, N.J.) measure Kd or Kd value with immobilized antigens c M5 chip with about 10 response units (RU).Letter speech It, according to description N-ethyl-N '-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and the N-of supplier N-Hydroxysuccinimide (NHS) activation carboxy methylation dextran biosensor matrix chip (CM5,Inc.).With 10mM sodium acetate, pH 4.8 dilutes capture antibodies (the most anti-human Fc), injects with the flow velocity of 30 μ l/min afterwards, and with anti- The further immobilization of Siglec-8 antibody.For kinetic measurement, inject dimer in 25 DEG C with the flow velocity of about 25 μ l/min Siglec-8 twice serial dilution in the PBS (PBST) containing 0.05%Tween 20.Use simple Lang Gemiao one to one That (Langmuir) combination model (Evaluation Software version 3.2) tied by matching simultaneously Close and the sensing figure that dissociates comes calculations incorporated speed (kon) and dissociation rate (koff).Dissociate carrying out calculated equilibrium than koff/kon Constant (Kd).See for example Chen, Y., et al., (1999) J.Mol.Biol.293:865-881.

In another embodiment, it is possible to use biosphere interferometric method measure anti-Siglec-8 antibody for The affinity of Siglec-8.In a kind of exemplary algoscopy, the Siglec-8 protein immobilization of band Fc label is caught to anti-human Catch on sensor, and with the mice of increasing concentration, chimeric, or humanization anti-Siglec-8Fab fragment incubation together, all to use Measure as the instruments such as such as Octet Red 384System (ForteBio) obtain affinity.

Such as, the binding affinity of anti-Siglec-8 antibody can also pass through Munson et al., Anal.Biochem., Scatchard described in 107:220 (1980) analyzes and uses standard technique known to association area to measure.Referring also to Scatchard,G.,Ann.N.Y.Acad.Sci.51:660(1947)。

2. antibody affinity

In one embodiment, the binding affinity of anti-Siglec-8 antibody can be surveyed by surface plasmon resonance The method of determining measures.It is, for example possible to use BIAcore T100 measures Kd or Kd value.Immobilization capture antibodies on CM5 chip (such as Goat anti human Fc and goat anti-mouse Fc).Can be with anti-human or anti-mouse antibody immobilization flow chamber.In certain temperature It is measured method, such as in 25 DEG C with the flow velocity of 30 μ l/min with certain flow velocity.Dilute in measuring buffer with multiple concentration Dimer Siglec-8, such as scope are the concentration of 15nM to 1.88pM.Capture antibodies, and carry out high efficient injection and penetrate, then dissociate. Flow chamber is regenerated with such as 50mM glycine pH 1.5 buffer such as grade.With reference to room and buffer injection is repeatedly measured as sky using sky In vain, and by 1:1 overall situation fitting parameter analysis result.

3. competition assay

Competition assay can be used to measure two kinds of antibody whether by identifying the most identical or that space is overlapping epi-position In conjunction with identical epi-position or a kind of another kind of antibodies bind antigen of antibody competition suppression.These algoscopys are known in the art. Typically, immobilized antigen or antigen-expressing cells on porous plate, and measure the marked antibodies of unmarked antibody blocking Ability.The conventional label of this type of competition assay is radioactive marker or enzyme marker.In some embodiments, originally The described anti-Siglec-8 antibody of literary composition is combined the cell table of a kind of cell (such as eosinocyte) with 2E2 antibody competition described herein Epi-position present on face.In some embodiments, anti-Siglec-8 antibody described herein with comprise SEQ ID NO:1's The heavy-chain variable domains of aminoacid sequence is competing with the antibody of the variable region of light chain of the aminoacid sequence comprising SEQ ID NO:15 Strive epi-position present on the cell surface of a kind of cell (such as eosinocyte) of combination.In some embodiments, institute herein State anti-Siglec-8 antibody to be combined with 2C4 antibody competition described herein on the cell surface of a kind of cell (such as eosinocyte) The epi-position existed.In some embodiments, anti-Siglec-8 antibody described herein with comprise SEQ ID NO:2 (such as U.S. State's patent No.8,207,305 finds) the heavy-chain variable domains of aminoacid sequence and comprise SEQ ID NO:4 (such as U.S. State's patent No.8,207,305 finds) the antibody competition of variable region of light chain of aminoacid sequence combine a kind of cell (example Such as eosinocyte) cell surface present on epi-position.

4. heat stability

In some respects, anti-Siglec-8 described herein has at least about 70 DEG C in hot transfer assay method, and at least about 71 DEG C, or the melting temperature (Tm) of at least about 72 DEG C.In a kind of exemplary thermal transfer assay method, will bag in qPCR thermal cycler Sample containing humanization anti-Siglec-8 antibody incubation 71 circulation, each circulation together with fluorescent dye (Sypro Orange) Raise 1 DEG C, to measure Tm.In some embodiments in this article, anti-Siglec-8 antibody have with mice 2E2 antibody and/ Or mice 2C4 antibody compares similar or higher Tm.In some embodiments in this article, anti-Siglec-8 antibody comprises bag Variable region of heavy chain containing the aminoacid sequence of SEQ ID NO:6；And/or comprise the aminoacid sequence selected from SEQ ID NO:16 or 21 The variable region of light chain of row.In some embodiments, anti-Siglec-8 antibody has compared with chimeric 2C4 antibody same or higher Tm.In some embodiments, anti-Siglec-8 antibody has and has the aminoacid sequence that comprises SEQ ID NO:84 Heavy chain compares same or higher Tm with the antibody of the light chain of the aminoacid sequence comprising SEQ ID NO:85.

5. biological activity assavs

In some respects, anti-Siglec-8 described herein induces eosinocyte apoptosis.In some other sides, institute herein State anti-Siglec-8 and cut down mastocyte.It is to it is known in the art that such as to join egg with film for assessing apoptotic algoscopy White V dyeing and TUNNEL algoscopy.In a kind of exemplary apoptosis algoscopy, resuspension carrys out autoblood sample in the medium The fresh buffy coat of product, and distribute in 96 hole U base plates.By a series of continuous 5 times of diluents of anti-Siglec-8 antibody Add to each hole, and by plate in 37 DEG C, 5%CO₂Incubation was more than 4 hours.The paraformaldehyde being used in PBS dilution is fixing thin Born of the same parents, and dye with conjugation of antibodies specific to eosinocyte, it is used for using microscopical detection.To at anti-Siglec-8 In the presence of antibody, the buffy coat of incubation assesses the eosinocyte group in total peripheral blood leucocyte, and not at anti-Siglec-8 In the presence of antibody, the buffy coat of incubation compares.In another kind of exemplary algoscopy, resuspension autoblood sample in the medium The eosinocyte (such as Miltenyi eosinocyte separating kit) of product purification, and exist at IL-5 or cultivate under disappearance Overnight.The eosinocyte cultivated by harvested by centrifugation subsequently, in the medium resuspension, and distribute in 96 hole U base plates.Will A series of continuous 5 times of diluents of anti-Siglec-8 antibody add to each hole, and by plate in 37 DEG C, 5%CO₂Incubation is more than 4 Hour.Use standard technique well known in the art, fixing cell, and dye with Annexin-V.Use microscopic examination addicted to eosin The number of cell.The cell of the purification of incubation in the presence of anti-Siglec-8 antibody is assessed the eosinocyte in sample Group, with not in the presence of anti-Siglec-8 antibody the cell of the purification of incubation compare.

In some respects, anti-Siglec-8 antibody induction ADCC activity described herein.In some other sides, described herein Anti-Siglec-8 antibody kills the mastocyte expressing Siglec-8 by ADCC activity.In some embodiments, compositions Comprise (i.e. without fucosylation) anti-Siglec-8 antibody of non-fucosylation.In some embodiments, with comprise portion The compositions dividing the anti-Siglec-8 antibody of fucosylation is compared, and comprises the described herein anti-Siglec-8 of non-fucosylation The compositions of antibody strengthens ADCC activity.It is well known in the art for assessing the algoscopy of ADCC activity and retouches herein State.In a kind of exemplary algoscopy, effector lymphocyte and target cell is used to measure ADCC activity.The example of effector lymphocyte includes NK cell (NK) cell, large granular lymphocyte (LGL), killing (LAK) cell of lymphokineactivation and comprise NK and LGL PBMC, or there is on cell surface the leukocyte of Fc receptor, such as neutrophil(e) cell, eosinocyte and macrophage. Target cell is any cell expressing the antigen that antibody to be assessed can identify on cell surface.A kind of example of this type of target cell Son is the eosinocyte expressing Siglec-8 on cell surface.The another kind of example of this type of target cell is on cell surface Express the mastocyte of Siglec-8.The reagent labels targets cell detected is dissolved with making cell.Reagent for labelling Example include radioactive substance, such as sodium chromate (Na₂ ⁵¹CrO₄).See for example Immunology, 14,181 (1968)； J.Immunol.Methods,172,227(1994)；And J.Immunol.Methods, 184,29 (1995).

In some respects, the activity of anti-Siglec-8 antibody suppression mast cell mediated described herein.Use hypertrophy Cell tryptase is as total Mast Cells and the biomarker of activation.For example, it is possible to measure in blood or urine Total and activated trypsinlike enzyme and histamine, N-.alpha.-Methylhistamine, and 11-β-prostaglandin F2 is to assess mastocyte Minimizing.A kind of exemplary mast cell activity algoscopy see for example the United States Patent (USP) Shen of publication number US 20110293631 Please.The algoscopy described in embodiment 2 herein can also be used to assess the anti-Siglec-8 antibody ADCC to mastocyte And apoptosis activity.

6. fusion protein binding assay

The binding assay carried out with fusion protein can be used to measure the epi-position by antibody recognition.Use and merge egg The algoscopy carrying out epitope mapping in vain is known in the art.Such as, on porous plate, immobilization comprises fusion the pure man Ig-Fc's The fusion protein of part Siglec-8 albumen, and measure the ability of antibodies fusion protein.In some embodiments, herein Described anti-Siglec-8 antibodies comprises the fusion protein of the aminoacid sequence of SEQ ID NO:115.In some embodiments In, anti-Siglec-8 antibodies described herein comprises the fusion protein of the aminoacid sequence of SEQ ID NO:116.Real at some Executing in scheme, anti-Siglec-8 antibodies described herein comprises the fusion protein of the aminoacid sequence of SEQ ID NO:117.? In some embodiments, anti-Siglec-8 antibodies described herein comprises the fusion of the aminoacid sequence of SEQ ID NO:118 Albumen.

III. prepared by antibody

Antibody described herein uses in this area to be prepared for generating the techniques available of antibody, and following each joint is specifically Describe its exemplary methods.

1. antibody fragment

The present invention contains antibody fragment.Antibody fragment can be generated by traditional means, such as enzymatic digestion, or logical Cross recombinant technique to generate.In some cases, antibody fragment is used to have superiority rather than complete antibody.Some antibody fragment Summary see Hudson et al. (2003) Nat.Med.9:129-134.

Have been developed for the multiple technologies for generating antibody fragment.Traditionally, by proteolytic digestion complete antibody Derive these fragments and (see for example Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117(1992)；And Brennan et al., Science 229:81 (1985)).But, now can be straight Connect and generated these fragments by recombinant host cell.Fab, Fv and scFv antibody fragment all can be at expression in escherichia coli with by large intestine Bacillus secretes, and so allows and easily produces these fragments substantial amounts of.Can separate anti-from phage antibody library discussed above Body fragment.Or, directly can reclaim Fab '-SH fragment chemical coupling to form F (ab ') from escherichia coli₂Fragment (Carter et al.,Bio/Technology 10:163-167(1992)).According to another kind of method, can directly train from recombinant host cell Support thing and separate F (ab ')₂Fragment.Comprise salvage receptor binding epitope residue, there is Fab and F of the Half-life in vivo of prolongation (ab′)₂Fragment is recorded in United States Patent (USP) No.5,869,046.For generating other technology of antibody fragment for the people that skillfully obtains employment Member can be apparent from.In certain embodiments, antibody is Single-Chain Fv Fragment of Murine (scFv).See WO 93/16185；The U.S. Patent No.5,571,894；And 5,587,458.Fv and scFv is to have entire binding site, lacks the unique type of constant region； So, they reduce non-specific binding when can be adapted to use in vivo.ScFv fusion protein can be built to generate effector The protein amino at scFv or the fusion of carboxyl terminal.See Antibody Engineering, Borrebaeck compile, see on Literary composition.Antibody fragment can also is that " linear antibodies ", such as such as United States Patent (USP) No.5, described in 641,870.This type of linearly resists Body can be monospecific or bispecific.

2. humanized antibody

The present invention contains humanized antibody.The multiple method for humanizing non-human antibodies is known in this area.Such as, people source Change antibody and can have one or more amino acid residue introduced from inhuman source.These non-human amino acid residues are usually referred to as " inputting " residue, they are normally taken from " input " variable domain.The method that substantially can follow Winter carries out humanization (Jones et al.(1986)Nature 321:522-525；Riechmann et al.(1988)Nature 332:323-327； Verhoeyen et al. (1988) Science239:1534-1536), the corresponding sequence of people's antibody is i.e. substituted with hypervariable region sequence Row.Therefore, this type of " humanization " antibody is chimeric antibody (United States Patent (USP) No.4,816,567), wherein considerably less than complete people The variable domain corresponding sequence of non-human species substitutes.In practice, humanized antibody is typically such as servant's antibody, and some of which is high Become district's residue to substitute with the residue in some possible similar site of FR residue rodent antibodies.

The selection of people's light chain and heavy chains's variable domain for preparing humanized antibody is probably for reducing antigenicity Important.According to so-called " the suitableeest (best-fit) " method, with the variable domain sequence pair of Rodents (such as mice) antibody Know that the whole library of people's variable domain sequence is screened.Then select with the immediate human sequence of Rodents as humanized antibody People's framework (Sims et al. (1993) J.Immunol.151:2296；Chothia et al.(1987) J.Mol.Biol.196:901).Another kind of method uses by the consensus sequence of everyone antibody of specific light chain or heavy chain subgroup Derivative specific frame.Identical frames can be used for several different humanized antibodies (Carter et al. (1992) Proc.Natl.Acad.Sci.USA 89:4285；Presta et al.(1993)J.Immunol.151:2623).

It is general it is also desirable that antibody retains the high-affinity to antigen and other favourable biological characteristics after humanization Property.In order to reach this purpose, according to a kind of method, by using the threedimensional model of parental array and humanized sequence to analyze parent The process of sequence and various conceptual humanized products prepares humanized antibody.Generally can adaptive immune globulin three-dimensional mould Type, this is familiar to those skilled in the art.Also can obtain diagram and the possibility of the selected candidate immunoglobulin sequences sequence of display The computer program of three-dimensional conformation structure.By checking that these display images allow that analysis residue is in candidate immunoglobulin sequences sequence May act in function, i.e. analyzing influence candidate immunoglobulin sequences combine the residue of the ability of its antigen.As such, it is possible to From receptor sequence and list entries, select FR residue and combine, thus obtaining desired antibody characteristic, such as to target antigen Affinity raises.It is said that in general, some hypervariable region residues directly and the most substantially relates to the impact combining antigen.

3. people's antibody

Selected from people's charon phages, people's anti-Siglec-8 antibody of the present invention can show that the Fv clone in storehouse can by combination Domain sequence builds with known people's constant domain sequence.Or, people's list of the present invention can be generated by hybridoma method Clone anti-Siglec-8 antibody.For having generated the human myeloma of human monoclonal antibodies and mice-people's heteromyeloma cell lines On the books, such as Kozbor, J.Immunol., 133:3001 (1984)；Brodeur et al.,Monoclonal Antibody Production Techniques and Applications,pp.51-63(Marcel Dekker,Inc., New York,1987)；And Boerner et al., J.Immunol., 147:86 (1991).

Likely generate lacking in the case of endogenous immunoglobulin generates that can to give birth to human antibodies after immunity complete The transgenic animal (such as mice) of complete or collected works.Such as, antibody heavy chain joining region in chimeric and germ line mutant mice has been described (JH) deletion of isozygotying of gene causes completely inhibiting of endogenous antibody tormation.A large amount of ethnic group is shifted in this type of germ line mutant mice It is that immunoglobulin gene can cause raw human antibodies after antigen is attacked.See for example Jakobovits et al., Proc.Natl.Acad.Sci.USA 90:2551(1993)；Jakobovits et al.,Nature 362:255(1993)； Bruggermann et al.,Year in Immunol.7:33(1993)。

Gene shuffling can also be used for deriving people's antibody from inhuman (such as Rodents) antibody, and wherein people's antibody has with initial Affinity that non-human antibody is similar and specificity.According to the method, it is also referred to as " the epi-position marking " (epitope Imprinting), the heavy chain of the non-human antibody fragment obtained by display technique of bacteriophage as described herein or variable region of light chain Employment V structure domain gene complete or collected works replace, and produce non-human chain/human chain scFv or Fab block polymer group.The selection carried out with antigen causes Following non-human chain/human chain is fitted together to the separation of scFv or Fab, and wherein human chain eliminates the most non-in one-level phage display is cloned Recover antigen binding site, i.e. epi-position after human chain and determine the selection of human chain spouse.Inhuman to replace residue repeating this process During chain, obtain people's antibody (seeing PCT WO 93/06213, be published on April 1st, 1993).Entered by CDR grafting with traditional The humanization of the non-human antibody of row is different, and this technology provides the antibody of complete people, and they FR or CDR without non-human origins are residual Base.

4. bi-specific antibody

Bi-specific antibody refers to the monoclonal antibody at least two not synantigen with binding specificity.Implement at some In scheme, bi-specific antibody is people's antibody or humanized antibody.In certain embodiments, one of binding specificity for Siglec-8, another of binding specificity is for other antigen any.In certain embodiments, bi-specific antibody can be in conjunction with Two kinds of different epi-positions of Siglec-8.Bi-specific antibody can be additionally used in and is positioned cytotoxic agent to express the thin of Siglec-8 Born of the same parents.Bi-specific antibody can be prepared as full length antibody or antibody fragment (such as F (ab ')₂Bi-specific antibody).

For build the method for bi-specific antibody be known in the art (see Millstein and Cuello, Nature 305:537(1983)；WO 93/08829, is published on May 13rd, 1993；And Traunecker et al., EMBO J.10:3655(1991)).Suresh et al. is see for example about the further details generating bi-specific antibody, Methods in Enzymology 121:210(1986).Bi-specific antibody includes crosslinking or " Heteroconjugate " antibody.Example As, a kind of antibody in heteroconjugate can be with affinity element coupling, another kind of antibody and biotin coupling.Can use any just The cross-linking method of profit prepares Heteroconjugate antibodies.Suitably cross-linking agent is well-known in the art, cross-links skill together with many Art is disclosed in United States Patent (USP) No.4,676,980 together.

5. single domain antibody

In some embodiment, the antibody of the present invention is single domain antibody (single-domain antibody).Single domain Antibody is the Single polypeptide chain of the heavy chain variable domain all or in part comprising antibody or light-chain variable domain all or in part.At certain In a little embodiments, single domain antibody is people single domain antibody (Domantis, Inc., Waltham, Mass.；See for example the U.S. special Profit No.6,248,516B1).In one embodiment, single domain antibody is made up of the heavy chain variable domain all or in part of antibody.

6. antibody variants

In some embodiment, contain the amino acid sequence modifications of antibody described herein.For example, it may be desirable to improve anti- The binding affinity of body and/or other biological characteristics.The amino acid sequence variation of antibody can be by by suitable change Introduce the nucleotide sequence of encoding antibody or prepared by peptide symthesis.This type of is modified in including such as antibody amino acids sequence Residue is deleted and/or inserts and/or substitutes.Any deletion can be carried out, insert and alternative combinations is to obtain final construction, if If final construction has desired feature.When preparing sequence, amino acid change can be introduced the aminoacid sequence of Subject antibodies Row.

Can be used for identifying in antibody has as some residue of preferred mutation position or the method in region that " Alanine-scanning lures Become ", as described in Cunningham and Wells (1989) Science 244:1081-1085.Here, a residue is identified Or one group of target residue (the most charged residue, such as arg, asp, his, lys, and glu) with neutral or electronegative Aminoacid (such as alanine or many alanine) is replaced, to affect the interaction of aminoacid and antigen.Then by or right Alternate site introduces more or other variant, weighs substituting the amino acid position showing function sensitive.Thus, although being used for The site introducing variant amino acid sequence predetermines, but the essence of sudden change itself need not predetermine.Such as, in order to Analyze the consequence specifying site sudden change, carry out Alanine-scanning or random mutagenesis at target codon or region, and to expressed Immunoglobulin screen desired activity.

Aminoacid sequence inserts and includes the fusion of amino and/or carboxyl terminal, length range by a residue to comprising one The polypeptide of hundred or more residues, and the sequence of single or multiple amino acid residue is inserted into.The example that end inserts includes tool There is the antibody of N end methionyl residue.Other insertion variant of antibody molecule includes anti-with enzyme or prolongation for N or the C end of antibody The peptide fusion of body serum half-life.

In certain embodiments, the antibody of the present invention there occurs and changes to improve or reduce the degree of antibody glycosylation. The glycosylation of polypeptide is typical or N-connection or O-connection.N-connection refers to that carbohydrate moiety is attached to Radix Asparagi acyl The side chain of amine residue.(wherein X is in addition to proline for tripeptide sequence asparagine-X-serine and asparagine-X-threonine Any aminoacid) it is the recognition sequence that carbohydrate moiety enzymatic is attached to asparagine side chain.So, in polypeptide this two The existence planting tripeptide sequence arbitrary creates potential glycosylation site.The glycosylation that O-connects refers to saccharide N-acetyl galactose Amine, one of galactose or xylose are attached to hydroxy-amino-acid, are most commonly that serine or threonine, but are used as 5-hydroxyl dried meat Propylhomoserin or 5-hydroxylysine.

Add in antibody or delete glycosylation site can by change aminoacid sequence thus create or eliminate one or Multiple above-mentioned tripeptide sequences and advantageously complete (for N-connect glycosylation site).Described change is also by original anti- The sequence of body is added, deletes, or substitute one or more serine or threonine residues carries out the (glycosyl connected for O- Change site).

If antibody comprises Fc district, then can change attachment carbohydrate thereon.Such as, U.S. Patent Application No. US The ripe carbohydrate structure having recorded shortage fucose in 2003/0157108 (Presta, L.) is attached to antibody Fc district Antibody.Referring also to US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.).WO 2003/011878 (Jean-Mairet etc.) and United States Patent (USP) No.6,602,684 (Umana etc.) refer to be attached to the carbon water in antibody Fc district Compound has the antibody of decile N-acetyl-glucosamine (GlcNAc).WO 1997/30087 (Patel etc.) reports in attachment The antibody of at least one galactose residue is had in the oligosaccharide in antibody Fc district.It is attached to its Fc district about there being change carbohydrate Antibody referring also to WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.).About having improvement glycosyl The antigen binding molecules changed is referring also to US 2005/0123546 (Umana etc.).

In certain embodiments, glycosylation variants comprises Fc district, and the carbohydrate structure being wherein attached to Fc district lacks Weary fucose or there is the fucose of minimizing.This type of variant has the ADCC function of improvement.Being optional that, Fc district is also included into one Step is improved at the one of ADCC or many places amino acid replacement, such as the replacement at Fc zone position 298,333 and/or 334 (compile by Eu residue Number mode).The example of the publication relating to " de-fucose type " or " fucose shortage type " antibody includes: US 2003/ 0157108；WO 2000/61739；WO 2001/29246；US 2003/0115614；US 2002/0164328；US 2004/ 0093621；US 2004/0132140；US 2004/0110704；US 2004/0110282；US 2004/0109865；WO 2003/085119；WO 2003/084570；WO 2005/035586；WO 2005/035778；WO 2005/053742； Okazaki et al.,J.Mol.Biol.336:1239-1249(2004)；Yamane-Ohnuki et al., Biotech.Bioeng.87:614(2004).The example of the cell line generating de-fucosylated antibody includes protein fucose Change Lec13CHO cell (the Ripka et al., Arch.Biochem.Biophys.249:533-545 (1986) of defect；The U.S. Number of patent application US 2003/0157108A1, Presta, L；And WO 2004/056312A1, Adams etc., especially embodiment 11) and knock out cell line, such as α-1, the Chinese hamster ovary celI (Yamane-Ohnuki that 6-fucosyl transferase gene FUT8 knocks out Et al., Biotech.Bioeng.87:614 (2004)) and process LAN β 1,4-N-acetyl glucosamine based transferase III And the cell of Gorky μ-mannosidase II (ManII) (GnT-III).

It is contemplated herein relative to the amount of fucose in the same antibody generated in wild-type CHO cells, there is reduction The antibody of fucose.Such as, if the amount of the fucose of this antibody (is such as generated by natural Chinese hamster ovary celI than in the case of other The Chinese hamster ovary celI of Natively glycosylated pattern, such as contains the Chinese hamster ovary celI of natural FUT8 gene) generate if the amount that had low. In certain embodiments, anti-Siglec-8 antibody provided herein is following antibody, on this antibody less than about 50%, The polysaccharide that the N of 40%, 30%, 20%, 10%, 5% or 1% connects comprises fucose.In certain embodiments, carry herein The anti-Siglec-8 antibody of confession is following antibody, and the polysaccharide that on this antibody, none N connects comprises fucose, i.e. this antibody is complete Do not have fucose or do not have fucose or non-fucosylation or without fucosylation.Can be by relative to attachment In the summation of all sugar structures (being such as combined, the structure with high mannose of heterozygosis) of Asn297, calculate sugar at Asn297 In chain, the average magnitude of fucose measures fucose amount, as measured by MALDI-TOF mass spectrometry, is the most such as recorded in WO 2008/077546.Asn297 refers to that the agedoite of the about the 297th (the Eu numbering of Fc district residue) being positioned in Fc district is residual Base；But, Asn297 can also be positioned at the 297th upstream or about ± 3, downstream ammonia owing to the minor sequence in antibody makes a variation Base acid, i.e. between the 294th and the 300th.In some embodiments, at least one or two heavy chains of antibody are non-rocks Algae is glycosylated.

In one embodiment, antibody has carried out changing to improve its serum half-life.In order to extend the serum of antibody Half-life, salvage receptor binding epitope can be mixed antibody (especially antibody fragment), such as such as United States Patent (USP) No.5,739, Described in 277.As used herein, term " salvage receptor binding epitope " refer to IgG molecule (such as IgG1, IgG2, IgG3, or IgG4) Fc district is responsible for extending the epi-position (US 2003/0190311 of serum half-life in IgG molecule body；The U.S. is special Profit No.6,821,505；United States Patent (USP) No.6,165,745；United States Patent (USP) No.5,624,821；United States Patent (USP) No.5,648, 260；United States Patent (USP) No.6,165,745；United States Patent (USP) No.5,834,597).

Another kind of variant is amino acid substitution variant.These variants have at least one amino acid residue to use in antibody molecule Different residues are replaced.The site interested carrying out substituting mutation includes hypervariable region, but is also covered by FR and changes.Table 1 " preferably replaces Generation " hurdle shows conservative replacement.If this type of replacement causes desired biologic activity to change, then can import in table 1 and be referred to as More substantial variations that is that " illustrate and substitute " or that further describe referring below to Amino Acid Classification, and screen product.

Table 1

Original Residue	Illustrate and substitute	Preferably substitute
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp；Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe；Nor-leucine	Leu
			Leu(L)	Nor-leucine；Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala；Nor-leucine	Leu

The substantive modification of antagonist biological characteristics can be by selecting the difference on effect to maintaining following aspect significant Substitute and realize: the structure of polypeptide backbone in (a) replacement area, such as, (fold) sheet or helical conformation, molecule at (b) target site Electric charge or hydrophobicity, or the volume of (c) side chain.According to the similarity of its side chain properties, aminoacid can be grouped as follows (A.L.Lehninger, in Biochemistry, second edition, pp.73-75, Worth Publishers, New York (1975)):

(1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

(2) uncharged, polarity: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)

(3) acid: Asp (D), Glu (E)

(4) alkalescence: Lys (K), Arg (R), His (H)

Or, according to common side chain properties, naturally occurring residue can be grouped as follows:

(1) hydrophobic: nor-leucine, Met, Ala, Val, Leu, Ile；

(2) neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln；

(3) acid: Asp, Glu；

(4) alkalescence: His, Lys, Arg；

(5) residue of chain orientation is affected: Gly, Pro；

(6) aromatic: Trp, Tyr, Phe.

Non-conservative replacement needs to replace another classification with the member of one of these classifications.It is all right that this type of substitutes residue Import conservative substitution sites, or import residue (non-conservative) site.

One or more hypervariable regions that one class alternative variations relates to substituting parental antibody (such as humanization or people's antibody) are residual Base.It is typically chosen the gained variant for exploitation further and can have change (such as relative to the parental antibody producing them Improve) biological characteristics.It is directed to use with the affine of phage display for generating a kind of facilitated method of this type of alternative variations Power is ripe.In brief, several hypervariable region sites (such as 6-7 site) are suddenlyd change, produce in each site all possible Amino acid replacement.The antibody display so generated is on filamentous phage particle, as the phage with each granule inner packing Coat protein (such as M13 gene III product) at least one of fusions.Then the variant to phage display screens its life Thing activity (such as binding affinity).In order to identify for the site, candidate hypervariable region modified, mutation can be scanned (such as Alanine-scanning) to identify the some hypervariable region residues that antigen combination is had significant contribution.Or it is multiple to analyze Ag-Ab The crystal structure of compound is to identify that the contact point between antibody and antigen is probably useful.This type of contact residues and neighbouring residue It is to carry out, according to techniques known in the art (including technology detailed in this article), the candidate locus that substitutes.Once produce such change Body, uses techniques known in the art (including the techniques described herein) to screen this group variant, may select a kind of or Multiple relevant assay has the antibody of good characteristic for further exploitation.

The nucleic acid molecules of encoding antibody amino acid sequence variation can be prepared by the multiple method that this area is known.These Method includes but not limited to separate (naturally occurring in the case of amino acid sequence variation) from natural origin, or by relatively Early the variant of preparation or the antibody of non-variant pattern carry out oligonucleotide mediated (or fixed point) mutation, and PCR mutation and boxlike lure Become and prepare.

May want to introduce at one in the Fc district of antibody of the present invention or many places are amino acid modified, thus generate Fc district and become Body.Fc region variants may be included in one or more amino acid position (including the position of hinge cysteine) and comprises amino acid modified People's Fc region sequence (such as human IgG1, IgG2, IgG3 or IgG4Fc district) of (such as substituting).In some embodiments, Fc district Variant comprises human IgG 4Fc district.In a further embodiment, this human IgG 4Fc district comprises amino acid replacement S228P, Wherein amino acid residue is according to the EU index number in Kabat.

Describe according to this and the teaching of this area, contain in some embodiment, the antibody of the present invention and wild type pair Answer antibody to compare and can comprise at one or many places change (in such as Fc district).Compared with their wild type counterparts, these resist Body still can substantially retain the identical characteristics required for therapeutic efficiency.Such as, it is believed that can carry out causing C1q to tie in Fc district Close and/or CDC (CDC) changes some of (i.e. or strengthen or weaken) and changes, such as WO99/ Described in 51642.Referring also to the Duncan and Winter, Nature 322:738-40 that pay close attention to Fc other example of region variants (1988)；United States Patent (USP) No.5,648,260；United States Patent (USP) No.5,624,821；And WO94/29351.WO00/42072 (Presta) describe the combination to FcR improve or the antibody variants of reduction with WO 2004/056312 (Lowman).Bright at this Acknowledgement enters the content of these patent publications as reference.Referring also to Shields et al., J.Biol.Chem.9 (2): 6591-6604(2001).Increased Plasma Half-life and to neonatal Fc receptor (FcRn) (it is responsible for Maternal immunoglobulin G is transferred to fetus) The knot of (Guyer et al., J.Immunol.117:587 (1976) and Kim et al., J.Immunol.24:249 (1994)) The antibody closing improvement is recorded in US2005/0014934A1 (Hinton etc.).These antibody comprise have at one or many places improve Fc The Fc district of the replacement that district is combined with FcRn.Fc region amino acid sequence and the C1q binding ability with change are raised and lowered many Peptide variant is recorded in United States Patent (USP) No.6,194,551B1, WO99/51642.Clearly take in these patent publications at this Hold as reference.Referring also to Idusogie et al., J.Immunol.164:4178-4184 (2000).

7. carrier, host cell and recombination method

For the antibody of the recombinant production present invention, separate its nucleic acid of coding, and be inserted into replicable vector, be used for into One-step cloning (DNA cloning) or expression.Old process can be used to be readily separated the DNA of encoding antibody and check order (as used energy Enough oligonucleotide probes being combined with the gene specific of encoding antibody heavy and light chain).Available many carriers.The selection of carrier Depend in part on the host cell that will use.It is said that in general, host cell is protokaryon or eucaryon (typically mammal) Source.Will be appreciated that the constant region of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant region, And this type of constant region can obtain from anyone or animal species.

Use prokaryotic host cell generation antibody:

A) vector construction

Standard recombinant techniques can be used to obtain the polynucleotide sequence of code book invention antibody polypeptides component.Can be from antibody Cellulation such as hybridoma separates desired polynucleotide sequence and checks order.Or, can use nucleotide synthesizer or Round pcr synthetic polyribonucleotides.Once obtain, the sequence of coded polypeptide is inserted and can replicate in prokaryotic hosts and express different The recombinant vector of source polynucleotide.For the present invention, the many carriers that this area can be used obtainable and know.Appropriate carrier Selection will primarily depend upon will insertion vector nucleic acid size and will be with the concrete host cell of vector.According to Its function (expand or expressing heterologous polynucleotide, or the two is furthermore) and the phase with its most resident concrete host cell thereof Capacitive, every kind of carrier contains multiple component.Support element typically includes, but not limited to: origin of replication, selected marker gene, starts Son, ribosome binding site (RBS), signal sequence, heterologous nucleic acids Insert Fragment, and transcription terminator.

It is said that in general, comprise the replicon derived from species compatible with host cell and control the plasmid vector of sequence and this A little hosts are used together.Carrier generally carries replication site, and can provide the mark sequence of Phenotypic Selection in converting cell Row.Such as, generally escherichia coli are converted with the pBR322 plasmid derived from species Escherichia coli.It is blue or green that pBR322 comprises coding ammonia benzyl Mycin (Amp) and the gene of tetracycline (Tet) resistance, thus provide the means of light identification of transformed cell.PBR322, it derives Thing, or other microorganism plasmid or phage also can comprise or modified and comprise can be endogenous for expressing by microorganism organism The promoter of protein.Carter et al., United States Patent (USP) No.5, describe in detail for expressing specific antibodies in 648,237 The example of pBR322 derivant.

It addition, by comprising the replicon compatible with host microorganism and the phage vector of sequence can be controlled as these places Main conversion carrier.Such as, phage such as λ GEM.TM.-11 can be used to build can be used for converting susceptible host cell such as The recombinant vector of escherichia coli LE392.

The expression vector of the present invention can comprise two to or multipair promoter-cistron, they encode each polypeptide component. Promoter is in the untranslated regulating and controlling sequence of cistron upstream (5 '), the expression of its regulation and control cistron.Prokaryotic promoter is usual Be divided into two classes, induction type and composition.Inducible promoter refer to respond condition of culture change (as nutrient existence with No or variations in temperature) and start the promoter that the elevated levels of the cistron being controlled is transcribed.

It is known that by a large amount of promoteres of multiple potential host cell identification.Source DNA is cut by limiting enzymic digestion In promoter and by separate promoter sequence insert the present invention carrier, thus can by select promoter with coding light chain Or the cistron DNA of heavy chain is operatively connected.Native promoter sequence and many allogeneic promoters can be used in instructing target gene Amplification and/or expression.In some embodiment, use allogeneic promoter, because compared with native target polypeptide promoter, it Generally allow for the higher of expressed target gene and transcribe and higher yield.

The promoter being applicable to prokaryotic hosts includes PhoA promoter, beta galactosidase and lactose promoter system, color Propylhomoserin (trp) promoter systems, and hybrid promoter such as tac or trc promoter.But, antibacterial has other of function Promoter (such as antibacterial known to other or phage promoter) is also suitable.Their nucleotide sequence has been delivered, Thus skilled work personnel can use provides the joint of any required restriction site or adapter by them and to encode target light chain (Siebenlist et al., Cell 20:269 (1980)) it is operatively connected with the cistron of heavy chain.

In one aspect of the invention, each cistron in recombinant vector comprises the expressed polypeptide of guidance and wears film transposition Secretory signal sequence component.It is said that in general, signal sequence can be the component of carrier, or it can be the target of insertion vector A part for polypeptid DNA.The signal sequence selected for the present invention should be affected by host cell identification processing (i.e. to be believed The excision of number peptidase) signal sequence.For nonrecognition the prokaryotic host cell of the signal sequences native processing heterologous polypeptide, will The signal sequence prokaryotic signal sequence selected from such as lower group substitutes: alkali phosphatase, penicillinase, Ipp, or heat-staple intestinal Toxin II (STII) targeting sequencing, LamB, PhoE, PelB, OmpA and MBP.In one embodiment of the invention, system is expressed The signal sequence all used in two cistron of system is STII signal sequence or its variant.

On the other hand, can occur in the Cytoplasm of host cell according to the generation of the immunoglobulin of the present invention, because of This need not there is secretory signal sequence in each cistron.In that, light chain immunoglobulin and heavy chain are at Cytoplasm Interior expression, folds and assembles and form Functional immunoglobulin.Some host strain (such as escherichia coli trxB-bacterial strain) provides Be conducive to the Cytoplasm condition of disulfide formation, thus allow that the correct of expressed protein subunit folds and assembling.Proba and Pluckthun,Gene 159:203(1995)。

The antibody of the present invention it be also possible to use the system of being expressed as and generates, and wherein the quantity ratios of expressed polypeptide component can To be regulated and controled, thus by secretion and the maximum production of the antibody of the present invention of correct assembling.This regulation and control are the most logical Realize after the translation intensity of regulation and control polypeptide component simultaneously.

Simmons et al., United States Patent (USP) No.5, disclose a kind of technology for regulating and controlling translation intensity in 840,523.It The variant of Translation initiator (TIR) is utilized in cistron.For specifying TIR, can create and there is certain limit translation intensity A series of aminoacid or Nucleic acid sequence variants, thus provide what the expectation expression for specific chain adjusted this factor to facilitate hands Section.The codon change that can change aminoacid sequence can be caused to generate TIR variant by conventional mutagenesis techniques.Implement at some In scheme, the change in nucleotide sequence is reticent.Change in TIR can include the number of such as Shine-Dalgarno sequence Mesh or the change of spacing, and the change in signal sequence.It is at code sequence for generating a kind of method of mutant signal sequences The beginning of row generates " password word bank " (i.e. change is reticent) not changing signal sequence aminoacid sequence.This can be by changing 3rd nucleotide position of each codon realizes；It addition, some aminoacid, such as leucine, serine, and essence ammonia Acid, has multiple first and second position, and this can increase complexity in building storehouse.Yansura et al.,METHODS:A Companion to Methods in Enzymol.4:151-158 (1992) describes this method of mutagenesis in detail.

In one embodiment, for each cistron in carrier, a group with certain limit TIR intensity is generated Carrier.This finite aggregate provides the expression of every chain and the yield of expectation antibody products in various TIR intensity groups Comparison under Heing.TIR intensity, Simmons et al., United States Patent (USP) can be measured by quantifying the expression of reporter gene No.5,840,523 has a detailed description.According to the comparison of translation intensity, desired indivedual TIR is selected to carry in the expression of the present invention Body construction is combined.

The prokaryotic host cell being suitable to express antibody of the present invention includes archeobacteria (Archaebacteria) and eubacteria (Eubacteria), such as Gram-negative or gram-positive organism.The example of useful antibacterial includes Escherichia (Escherichia) (such as colon bacillus E.coli), bacillus (Bacillus) is (such as bacillus subtilis B.subtilis), Enterobacter (Enterobacteria), Rhodopseudomonas (Pseudomonas) is (such as Pseudomonas aeruginosa P.aeruginosa) species, Salmonella typhimurium (Salmonella typhimurium), serratia marcescens (Serratia marcescans), Klebsiella (Klebsiella), proteus (Proteus), Shigella (Shigella), rhizobium (Rhizobium), Vitreoscilla (Vitreoscilla), or paracoccus (Paracoccus).In one embodiment, gram-negative cells is used.In one embodiment, large intestine bar is used Bacterium cell is as the host of the present invention.The example of coli strain includes bacterial strain W3110 (Bachmann, Cellular and Molecular Biology, volume 2, Washington, D.C., American Academy Of Microbiology, 1987, the 1190-1219 page； ATCC preserving number 27,325) and derivant, including having genotype W3110 Δ fhuA (Δ tonA) ptr3 lac Iq The bacterial strain 33D3 (United States Patent (USP) No.5,639,635) of lacL8 Δ ompT Δ (nmpc-fepE) degP41 kanR.Other bacterial strain And derivant, such as escherichia coli 294 (ATCC 31,446), escherichia coli B, escherichia coli λ 1776 (ATCC 31,537) Also it is suitable with escherichia coli RV308 (ATCC 31,608).These examples simply illustrate and unrestricted.Use is known in this area In building the method with any of above bacterial derivation thing specifying genotype, see for example Bass et al., Proteins 8:309-314(1990).Typically required consideration replicon reproducibility in bacterial cell selects suitable antibacterial.Example As, when such as pBR322, pBR325, pACYC177 or pKN410 provide replicon to the well-known plasmid of use, large intestine Bacillus, Serratia, or Salmonella ssp may be suitable for use as host.Generally, host cell should secrete minimum Proteolytic enzyme, and may want to cell cultivate in mix extra protease inhibitor.

B) antibody tormation

With above-mentioned expression vector transformed host cell, and for evoked promoter, select transformant or amplification coding phase The conventional nutrient culture hoping the gene of sequence and suitably change is cultivated.

Conversion will import prokaryotic hosts by DNA so that DNA can replicate, or as extra-chromosomal element or Pass through chromosomal composition.According to host cell used, the standard technique being suitable to these cells is used to convert.Use calcium chloride Calcium treatment be generally used for the bacterial cell with firm cell-wall barriers.Another kind of method for transformation uses Polyethylene Glycol/DMSO. A kind of technology that also has used is electroporation.

This area know and be suitable to cultivate selected host cell culture medium in cultivate for generating polypeptide of the present invention Prokaryotic cell.The example of suitable culture medium includes the LB culture medium (Luria broth) that with the addition of required nutritional supplement.? In some embodiment, the selective agent that culture medium selects possibly together with the structure according to expression vector, allow with selectivity and comprise The prokaryotic cell growth of expression vector.Such as, in the culture medium for cultivating the cell expressing ampicillin resistance gene Add ampicillin.

Beyond carbon, nitrogen, and inorganic phosphate sources, also can be containing any required fill-in of debita spissitudo or single Solely add or as with another kind of fill-in or the mixture of culture medium, such as compound nitrogen source.Being optional that, culture medium can contain There are one or more reducing agents being selected from lower group: glutathion, cysteine, cystamine, thioglycolate salt/ester, two sulfur erythroses Alcohol and dithiothreitol, DTT.

Prokaryotic host cell is cultivated in suitable temperature.In certain embodiments, for cultivating escherichia coli, cultivation Temperature range is about 20 DEG C to about 39 DEG C, about 25 DEG C to about 37 DEG C, or about 30 DEG C.Depend primarily on host organisms, culture medium PH can be any pH that scope is about 5 to about 9.In certain embodiments, for escherichia coli, pH is about 6.8 to about 7.4, or about 7.0.

If the expression vector of the present invention uses inducible promoter, then lure under conditions of being suitable to activate promoter Lead protein expression.In one aspect of the invention, PhoA promoter is used to control transcribing of polypeptide.Therefore, in order to induce, The host cell through converting is cultivated in phosphate limits culture medium.In certain embodiments, phosphate limits culture medium It is C.R.A.P culture medium (see for example Simmons et al., J.Immunol.Methods 263:133-147 (2002)). According to the vector construct used, other inducer multiple can be used, as is known in the art.

In one embodiment, expressed polypeptide of the present invention is secreted in the pericentral siphon of host cell and therefrom reclaims. Protein Recovery generally involves destruction microorganism, generally by such as osmotic shock (osmotic shock), and supersound process or split The means such as solution.Once cell is destroyed, can be by centrifugal or filtration clear cell debris or whole cell.Can be by such as Affine resin chromatography is further purified protein.Or, protein may be transported in culture fluid and therefrom separate.Can be from cultivation Liquid scavenger cell, and culture supernatants is filtered and concentrates, it is used for being further purified generated protein.Can use and generally know The method in road such as polyacrylamide gel electrophoresis (PAGE) and western blot are analyzed and are separated further and many expressed by qualification Peptide.

In one aspect of the invention, antibody producing is carried out in a large number by sweat.Multiple extensive fedbatch is sent out Ferment flow process can be used for producing recombiant protein.Large scale fermentation has the capacity of at least 1000 liters, is about in certain embodiments The capacity of 1,000 to 100,000 liters.These fermentation tanks use agitator paddle to distribute oxygen and nutrient, especially glucose.Little Scale fermentation is often referred to the fermentation carried out in the fermentation tank of volume capacity no more than about 100 liters, can range from about 1 and rises to about 100 liters.

During the fermentation, generally cell is cultivated under suitable conditions to expect density (such as OD550 about 180-220, It is in early days stable phase at this phase cell) start the induction of protein expression afterwards.According to the vector construct used, can make Use multiple inducer, that know as this area and above-described.Before induction, cell can be cultivated relatively short period of time.Generally By cell induction about 12-50 hour, but longer or shorter induction time can be used.

In order to improve yield and the quality of polypeptide of the present invention, multinomial fermentation condition can be revised.Such as, secreted in order to improve The correct assembling of antibody polypeptides and folding, the expression chaperone such as Dsb albumen that can overuse (DsbA, DsbB, DsbC, DsbD and/or DsbG) or the additional carrier of FkpA (there is a kind of peptidyl prolyl of Chaperone Activity-cis, trans-isomerase) Carry out cotransformation host prokaryotic cell.Have turned out chaperone and promote that the heterologous protein of generation is just in bacterial host cell Really fold and dissolubility.Chen et al.,J.Biol.Chem.274:19601-19605(1999)；Georgiou et al. is beautiful State's patent No.6,083,715；Georgiou et al., United States Patent (USP) No.6,027,888；Bothmann and Pluckthun, J.Biol.Chem.275:17100-17105(2000)；Ramm and Pluckthun,J.Biol.Chem.275:17106- 17113(2000)；Arie et al.,Mol.Microbiol.39:199-210(2001).

For the Proteolytic enzyme fall by expressed heterologous protein (especially sensitive to Proteolytic enzyme heterologous protein) To minimum, some host strain of Proteolytic enzyme enzyme defect can be used for the present invention.Such as, host cell strains can be modified, Encode and the gene of known bacterialprotease carries out genetic mutation, such as proteinase II I, OmpT, DegP, Tsp, protease I, egg White enzyme Mi, protease V, protease VI and combinations thereof.Some e. coli protein enzyme defect bacterial strain can be obtained, see for example Joly et al., (1998) see above；Georgiou et al., United States Patent (USP) No.5,264,365；Georgiou et al., the U.S. Patent No.5,508,192；Hara et al.,Microbial Drug Resistance 2:63-72(1996).

In one embodiment, in the expression system of the present invention, Proteolytic enzyme enzyme defect is used and through overexpression The coli strain of the Plastid transformation of one or more chaperones is as host cell.

C) antibody purification

In one embodiment, the antibody protein generated herein it is further purified to obtain the system of substantially homogeneity Product, for further measuring and using.The Standard protein purification method that this area is known can be used.Following flow process is to close The illustration of suitable purification flow process: immunity is affine or classification on ion exchange column, and ethanol precipitates, reversed-phase HPLC, tripoli or cation Chromatography on exchanger resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and use such as Sephadex G-75 Gel filtration.

In one aspect, the protein A being fixed in solid phase is used for the immunoaffinity purification of antibody products of the present invention.Albumen A is the 41kD cell wall protein from staphylococcus aureus (Staphylococcus aureas), and it is tied with high-affinity Close antibody Fc district.Lindmark et al.,J.Immunol.Meth.62:1-13(1983).The solid phase that protein A is fixed thereon can To be the pillar with glass or quartz surfaces, or controlled pore glass post or silicic acid post.In some applications, pillar is with all As the reagent such as glycerol are coated, in order to likely to prevent the non-specific adhesion of pollutant.

As the first step of purification, the prepared product derived from cell culture described above can be applied to protein A solid Surely change in solid phase so that purpose antibody Specific binding proteins A.Then solid phase is cleaned to remove the pollution with solid phase non-specific binding Thing.Purpose antibody is reclaimed from solid phase finally by eluting.

Use eukaryotic host cell generation antibody:

Carrier for eukaryotic host cell generally includes one or more following non-limiting components: signal sequence, multiple Starting point processed, one or more marker gene, enhancer element, promoter, and transcription terminator.

A) signal sequence component

The carrier used in eukaryotic host cell also can comprise signal sequence at the N end of purpose mature protein or polypeptide Or there is other polypeptide of special cleavage site.Can select by host cell identification and process (i.e. being excised by signal peptidase) Heterologous signal sequences.In mammalian cell expression, available mammalian signal sequences and viral secretory are leading, example Such as herpes simplex gD signal.The DNA of these prosomas is connected in the way of meeting reading frame the DNA of encoding antibody.

B) origin of replication

Generally, mammalian expression vector need not origin of replication component.Such as, SV40 starting point generally may be only because comprising Early promoter just uses.

C) Select gene component

Express and cloning vehicle can comprise Select gene, also referred to as selection marker.Typical Select gene coding is such as laid eggs White matter: (a) gives antibiotic or the resistance of other toxin, such as ampicillin, neomycin, methotrexate or tetracycline；(b) Supply corresponding auxotrophy；Or (c) provides the critical nutrients that can not obtain from complex medium.

One example of selection scheme utilizes medicine to block the growth of host cell.Through that of heterologous gene successful conversion A little Hemapoiesis give the protein of drug resistance, thus survive selection scheme.The example of this type of dominant selection uses medicine Neomycin, mycophenolic acid and hygromycin.

Another example of the selection marker being suitable to mammalian cell can identify picked-up antibody nucleic acids of having the ability The selection marker of cell, such as DHFR, thymidine kinase, metallothionein I and II, primate metallothionein's gene, adenosine takes off Ammonia enzyme, ODC Ornithine decarboxylase etc..

Such as, in some embodiment, first pass through all transformants containing methotrexate (the one of Mtx, DHFR Kind of competitive antagonist) culture medium in carry out cultivating and identify through the cell that DHFR Select gene converts.Some embodiment party In case, when using wild type DHFR, suitable host cell is Chinese hamster ovary (CHO) cell line of DHFR active defects (such as ATCC CRL-9096).

Or, can be by containing the selective agent such as aminoglycoside antibiotics such as kanamycin for selection marker, newly The culture medium of mycin or G418 is cultivated cell and selects encoded antibody, wild type DHFR protein, and another kind of selection marker The DNA sequence of such as aminoglycoside 3 '-phosphotransferase (APH) converts or the host cell of cotransformation (particularly comprises endogenous The wild type host of DHFR).See United States Patent (USP) No.4,965,199.Host cell can include NS0, CHOK1, CHOK1SV or Derivant, including the cell line of glutamine synthetase (GS) defect.Use GS as the selection marker of mammalian cell Method is recorded in United States Patent (USP) No.5,122,464 and United States Patent (USP) No.5,891,693.

D) promoter component

Express and cloning vehicle generally comprises the promoter by host organisms identification, and and encoding polypeptides of interest The nucleic acid of (such as antibody) is operatively connected.The promoter sequence of known genuine nucleus.Such as, it is true that all eukaryotic genes All having rich in AT district, it is positioned at about 25 to 30 bases of site upstream of initiation transcription.At the most polygenic transcriptional start point The another kind of sequence found at the base of 70 to 80, upstream is CNCAAT district, and wherein N can be any nucleotide.Most of true 3 ' ends of karyogene are AATAAA sequences, and it is probably the 3 ' ends to coded sequence and adds the signal of polyadenylic acid (polyA) tail. In certain embodiments, any or all these sequences can suitably be inserted in carrier for expression of eukaryon.

Mammalian host cell is transcribed by such as from virus (such as polyoma virus, fowlpox virus, gland from carrier Virus (such as 2 type adenovirus), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, With simian virus 40 (SV40)) genome acquisition, from heterologous mammal promoter (such as actin promoter or immunity Globin promoter), from the control of the promoter of heat-shock promoters, if these promoteres and host cell systems phase If appearance.

Form with SV40 restriction fragment obtains the early and late promoter of SV40 virus easily, and this fragment is also wrapped Containing SV40 virus origin of replication.The morning immediately of human cytomegalic inclusion disease virus is obtained easily with the form of HindIII E restriction fragment Phase promoter.United States Patent (USP) 4,419,446 discloses use bovine papilloma virus as carrier table in mammalian hosts Reach the system of DNA.United States Patent (USP) No.4,601,978 has been recorded a kind of amendment of this system.Coming about in mouse cell From the control following table intelligent beta-interferon cDNA of the thymidine kinase promoter of herpes simplex virus referring also to Reyes et al., Nature 297:598-601(1982).Or, rous sarcoma virus long terminal repeat can be used as promoter.

E) enhancer element component

Often through inserting enhancer sequence in the carrier to improve the higher eucaryotic cells DNA to code book invention antibody Transcribe.It is now know that from mammalian genes (globulin, elastoser, albumin, α-fetoprotein and insulin) Many enhancer sequence.But, generally use the enhancer from eukaryotic cell virus.Example includes SV40 origin of replication late period The enhancer (bp 100-270) of side, human cytomegalic inclusion disease virus early promoter enhancer, mouse cytomegalovirus early promoter Enhancer, the enhancer of polyoma virus origin of replication side in late period, and adenovirus cancers.About the enhancing activating eukaryotic promoter Sub-element is referring also to Yaniv, Nature 297:17-18 (1982).Enhancer can montage in carrier, be positioned at antibody polypeptides The 5 ' of coded sequence or 3 ' positions, but it is normally at 5 ' sites of promoter.

F) tanscription termination component

In eukaryotic host cell use expression vector also can comprise termination transcribe with stable mRNA necessary to sequence. This type of sequence generally can obtain with 3 ' ends once in a while from 5 ' ends of eucaryon or viral DNA or cDNA untranslated region.These regions are wrapped The untranslated region transcription of the mRNA being contained in encoding antibody becomes the nucleotide segment of polyadenylated fragments.A kind of useful transcribing Terminating component is bovine growth hormone polyadenylation district.See WO94/11026 and disclosed in expression vector.

G) selection of host cell and conversion

The host cell being suitable to the DNA in clone or expression carrier herein includes higher eucaryotic cells described herein, bag Include vertebrate host cell.Vertebrate cells breeding in cultivating (tissue culture) has become as old process.Useful The example of mammalian host cell line has monkey kidney CV1 system (COS-7, ATCC CRL 1651) converted through SV40, human embryo kidney (HEK) system (293 cells or 293 cells of sub-clone for suspension culture, Graham et al., J.Gen.Virol.36:59 (1977)), Baby hamster kidney cell (BHK, ATCC CCL 10), Chinese hamster ovary cell/-DHFR (CHO, Urlaub et al., Proc.Natl.Acad.Sci.USA 77:4216 (1980)), mice Sai Tuoli (Sertoli) cell (TM4, Mather, Biol.Reprod.23:243-251 (1980)), monkey-kidney cells (CV1, ATCC CCL 70), African green monkey kidney cell (VERO- 76, ATCC CRL 1587), human cervical carcinoma cell (HELA, ATCC CCL2), Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL 34), cattle Mus (buffalo rat) hepatocyte (BRL 3A, ATCC CRL 1442), human pneumonocyte (W138, ATCC CCL 75), people liver is thin Born of the same parents (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL 51), TRI cell (Mather et al., Annals N.Y.Acad.Sci.383:44-68 (1982)), MRC 5 cell, FS4 cell, CHOK1 cell, CHOK1SV cell Or derivatives thereof and human liver cell tumor (hepatoma) are (Hep G2).

In order to generate antibody, with expression mentioned above or cloning vehicle transformed host cell, and for evoked promoter, The conventional nutrient culture selecting transformant or the amplification coding expectation gene of sequence and suitably change is cultivated.

H) cultivation of host cell

The host cell for generating antibody of the present invention can be cultivated in multiple culture medium.Commercially available culture medium such as Ham Family name F10 (Sigma), minimum essential medium (MEM, Sigma), RPMI-1640 (Sigma), and DulbeccoShi improve Eagle Family name's culture medium (DMEM, Sigma) is suitable to cultivate host cell.It addition, any culture medium described in following documents can be used to make Culture medium for host cell: Ham et al., Meth.Enz.58:44 (1979)；Barnes et al., Anal.Biochem.102:255(1980)；United States Patent (USP) No.4,767,704；4,657,866；4,927,762；4,560, 655；Or 5,122,469；WO 90/03430；WO 87/00195；Or United States Patent (USP) review 30,985.These culture medium any can Supplementing hormone and/or other somatomedin (such as insulin, transferrin or epidermal growth factor) as required, salt is (such as Sodium chloride, calcium, magnesium and phosphate), buffer agent (such as HEPES), nucleotide (such as adenosine and thymidine), antibiotic is (such as GENTAMYCIN^TMMedicine), trace element (is defined as the inorganic compound generally existed with the final concentration of micro-molar range), and Glucose or the equivalence energy.Other fill-in any that those skilled in the art will know that can be contained with suitable concentration.Cultivate bar Part such as temperature, pH etc. is and expresses and the host cell that selects is previously used, and this is obvious for those of ordinary skill.

I) purification of antibody

When using recombinant technique, can be at intracellular generation antibody, or direct secretion is in culture medium.If at cell Interior generation antibody, then first can remove particle debris by such as centrifugal or ultrafiltration, or host cell or cracking sheet Section.If antibody-secreting is in culture medium, then can first by commercialization protein concentration filter (such as Amicon or Millipore Pellicon ultra filtration unit) concentrate the supernatant from these expression systems.Can wrap in any above-mentioned steps Include protease inhibitor such as PMSF to suppress Proteolytic enzyme, and can include that antibiotic is to prevent the growth of external contaminant.

Can use such as hydroxyapatite, gel electrophoresis, (affinity chromatograph is one skill easily with affinity chromatograph in dialysis Art) carry out the antibody compositions that purification is prepared from cell.Protein A depends on appointing present in antibody as the suitability of affinity ligand The kind of what immunoglobulin Fc domain and isotype.It is based on people γ 1, γ 2, or γ 4 heavy chain anti-that protein A can be used for purification Body (Lindmark et al., J.Immunol.Meth.62:1-13 (1983)).Protein G is recommended for all mouse isotypes With people γ 3 (Guss et al., EMBO J.5:1567-1575 (1986)).Substrate accompanying by affinity ligand can be agar Sugar, but other substrate can be used.The substrate of physically stable such as controlled pore glass or poly-(styrene divinyl) benzene can obtain Must flow velocity more faster than agarose and shorter process time.If antibody comprises CH3 domain, then can use Bakerbond ABX^TMResin (J.T.Baker, Phillipsburg, N.J.) is purified.According to antibody to be recycled, it is possible to use other egg Classification on white matter purification technique such as ion exchange column, ethanol precipitates, reversed-phase HPLC, the chromatography on tripoli, heparin SEPHAROSE^TMOn chromatography, the chromatography in anion or cation exchange resin (such as poly-aspartate post), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

After any any preliminary purification step, can the mixture containing purpose antibody and pollutant be carried out the purest Change, the lowest pH hydrophobic interaction chromatography, use the elution buffer of pH about 2.5-4.5, at low salt concn (such as from about 0- 0.25M salt) carry out.

It is said that in general, be used for preparing for research, the various methods of the antibody of test and Clinical practice are that this area is the most perfect Setting up, with method as described above be consistent and/or those skilled in the art think that for specific antibody interested be suitable Suitable.

The generation of non-defucosylated antibody

Method for preparing the antibody that fucosylation degree reduces provided herein.Such as, the side being contemplated herein Method includes but not limited to use cell line (such as Lec13CHO cell, α-1, the 6-fucose of protein fucosylation defect Based transferase gene knockout Chinese hamster ovary celI, process LAN β Isosorbide-5-Nitrae-N-acetyl glucosamine based transferase III and further process LAN are high The cell of you base μ-mannosidase II (ManII), etc.), and in the cell culture medium for generating antibody, add fucose Analog.See Ripka et al., Arch.Biochem.Biophys.249:533-545 (1986)；U.S. Patent application No.US 2003/0157108A1,Presta,L；WO 2004/056312A1；Yamane-Ohnuki et al., Biotech.Bioeng.87:614(2004)；With United States Patent (USP) No.8,574,907.Other for reducing antibody fucose content Technology include the Glymaxx technology described in U.S. Patent Application Publication text No.2012/0214975.Other for reducing The technology of the fucose content of antibody is additionally included in the cell culture medium for generate antibody adds one or more glycosidase Inhibitor.Glycosidase inhibitor includes alpha-glucosidase I, alpha-glucosidase II, and alpha-Mannosidase I.Real at some Executing in scheme, glycosidase inhibitor is the inhibitor (the most several husband's alkali) of alpha-Mannosidase I.

As used in this article, " core fucosylation " refers to that the reducing end under neutral of polysaccharide connected at N is to N-acetyl group Glycosamine (" GlcNAc ") adds fucose (" fucosylation ").Also provide for antibody of being generated by this type of method and combinations thereof Thing.

In some embodiments, the fucosylation of the sugar chain being bound to the compound N-glucosides connection in Fc district (or territory) is Reduce.As used in this article, " sugar chain that compound N-glucosides connects " is typically bonded to agedoite 297 (according to Kabat volume Number), although the sugar chain that compound N-glucosides connects can also be connected to other asparagine residue." the sugar that compound N-glucosides connects Chain " get rid of high mannose type sugar chain, wherein the non reducing end at core texture only mixes mannose, but includes 1) compound Type, wherein the non reducing end side of core texture has one or more galactose-N-acetyl glucosamine (also referred to as " gal-GlcNAc ") non reducing end of branch and Gal-GlcNAc optionally has sialic acid, two typing N-acetyl group glucose Amine, like this；Or 2) heterozygous, wherein the non reducing end side of core texture has the sugar that high mannose N-glucosides connects Chain and compound N-glucosides connect sugar chain Liang Zhong branch.

In some embodiments, " sugar chain that compound N-glucosides connects " includes compound, and wherein the non-of core texture is gone back Originality end side has zero, the branch of one or more galactose-N-acetyl glucosamines (also referred to as " gal-GlcNAc ") And the non reducing end side of Gal-GlcNAc has such as sialic acid the most further, two typing N-acetyl glucosamines, all Structure such as this type of.

According to this method, the sugar chain that compound N-glucosides connects generally only mixes indivisible fucose.Such as, various In embodiment, in compositions less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, few In about 15%, the antibody of less than about 10%, less than about 5%, or less than about 1% has the core fucose base caused by fucose Change.In some embodiments, substantially none (the most less than about 0.5%) antibody in compositions has core caused by fucose Fucosylation.In some embodiments, more than about 40% in compositions, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 91%, more than about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, or the antibody more than about 99% is non-fucosylation.

In some embodiments, following antibody provided herein, the most substantially none (the most less than about 0.5%) N- The carbohydrate chain that glucosides connects contains fucosyl residues.In some embodiments, following antibody provided herein, its Middle heavy chain of antibody at least one or two are non-fucosylations.

As set forth above, it is possible to utilize multiple mammalian hosts-expression vector system to express antibody.Some embodiment party In case, culture medium does not supplements fucose.In some embodiments, culture medium is added the fucose analogue of effective dose.? In this linguistic context, " effective dose " refers to analog to be enough to fucose to be incorporated to that antibody is combined the sugar chain of N-glucosides-connection and reduces at least about 10%, at least about 20%, at least about 30%, at least about 40% or the amount of at least about 50%.In some embodiments, with at rock The antibody that the lower host cell cultivated of algae sugar analogue disappearance generates is compared, and the antibody generated by this method is comprised at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50% non-core fucosylation albumen is (the most scarce Weary core fucosylation).

Such as can measure the N-second that is not bound in the reducing end under neutral of sugar chain of fucose as described in embodiment The content of the sugar chain of the N-acetyl glucosamine that fucose is bound in the reducing end under neutral of sugar chain by the sugar chain of acyl glucamides (such as ratio).Other method includes that hydrazinolysis or enzymic digestion (see for example Biochemical Experimentation Methods 23:Method for Studying Glycoprotein Sugar Chain(Japan Scientific Societies Press), edited by Reiko Takahashi (1989)), fluorescent labeling or labelled with radioisotope The sugar chain of release, then separates marked sugar chain by chromatography.Further, can survey by analyzing chain by HPAEC-PAD method The compositions (see for example J.Liq Chromatogr.6:1557 (1983)) of the sugar chain of fixed release is (referring generally to United States Patent (USP) Application disclosure No.2004/0110282).

IV. compositions

In some respects, compositions (the such as medicine that comprise described herein any anti-Siglec-8 antibody is also provided herein Compositions).In some respects, the compositions comprising anti-Siglec-8 antibody described herein provided herein, wherein this antibody Comprising Fc district and be connected to the carbohydrate chain that the N-glucosides in this Fc district connects, this N-glucosides of the most less than about 50% connects Carbohydrate chain contain fucosyl residues.In some respects, provided herein anti-Siglec-8 antibody described herein is comprised Compositions, wherein this antibody comprise Fc district and be connected to this Fc district N-glucosides connect carbohydrate chain, the most substantially The carbohydrate chain that none this N-glucosides upper connects contains fucosyl residues.

By having active component and optional pharmaceutical acceptable carrier, excipient or the stabilizer of expectation purity (Remington:The Science and Practice of Pharmacy, the 20th edition, Lippincott Williams& Wiklins, Pub., Gennaro compile, Philadelphia, Pa.2000) mix and prepare treatment preparaton, for storage.Can The carrier accepted, excipient or stabilizer are nontoxic in the dosage used and concentration to receiver, and include buffer agent； Antioxidant, including ascorbic acid, methionine, vitamin E, sodium metabisulfite；Preservative；Isoosmotic adjusting agent (isotonicifier)；Stabilizer；Metal composite, such as Zn-protein complex；Chelating agen, such as EDTA；And/or it is non- Ionic surface active agent.

Buffer agent can be used to control pH optimizing in the range of therapeutic efficiency, especially at stability dependency in pH's In situation.Buffer agent can be about the concentration existence of 50mM to about 250mM with scope.Be suitable to the buffering being used in conjunction with Agent includes organic acid and mineral acid and salt thereof, such as citrate, phosphate, succinate, tartrate, Fumaric acid Salt, gluconate, oxalates, lactate, acetate.It addition, buffer agent can be by histidine and front three amine salt such as Tris Constitute.

Preservative can be added to prevent growth of microorganism, and generally exist with the scope of about 0.2%-1% (w/v). The preservative being suitable to be used in conjunction with includes octadecyl dimethyl benzyl ammonium chloride；The double ammonium of chlorination hexane；Benzene pricks halogen Ammonium (benzene pricks iodine ammonium for such as benzalkonium chloride, benzalkonium bromide), benzethonium chloride；Thimerosal；Phenol, butanol or benzyl alcohol；Para hydroxybenzene first Acid hydrocarbyl carbonate, such as methyl parahydroxybenzoate or propyl ester；Catechol；Resorcinol；Hexalin；3-amylalcohol；And metacresol.

There is isotonic agent (being sometimes referred to as " stabilizer ") and with regulation or maintain the isotonicity of liquid in compositions.When with greatly , when charged biomolecule such as protein and antibody are used together, they are often referred to as " stabilizer ", because they energy Interact with the charged groups of amino acid side chain, thus alleviate intermolecular and intramolecular interaction gesture (potential).Isotonic agent can be with about 0.1% to about 25% (by weight), or any amount between about 1 to about 5% is deposited , it considers the relative quantity of other composition.In some embodiments, isotonic agent includes polyhydroxy sugar alcohol, triatomic Or the sugar alcohol of higher level, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol.

Other excipient includes the medicament that can undertake following one or more function: (1) filler (bulking agent)；(2) solubilizing agent；(3) stabilizer；(4) degeneration or the medicament of the adhesion to chamber wall are prevented.This type of excipient bag Include: polyhydroxy sugar alcohol (listed above)；Aminoacid, such as alanine, glycine, glutamine, agedoite, histidine, Arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine etc.；Organic sugar or sugar alcohol, such as sucrose, Lactose, lactose, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, muscle inose, Myo-Inositol, half Lactose, galactitol, glycerol, cyclitol (such as inositol), Polyethylene Glycol；The reducing agent of sulfur-bearing, such as carbamide, glutathion, Thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate；Low molecular weight protein, such as human serum Albumin, bovine serum albumin, gelatin or other immunoglobulin；Hydrophilic polymer, such as polyvinylpyrrolidone；Single Sugar, such as xylose, mannose, fructose, glucose；Disaccharide, such as lactose, maltose, sucrose；Trisaccharide, such as Raffinose；Many Sugar, such as dextrin or glucosan (dextran).

Can exist nonionic surfactant or detergent (also referred to as " wetting agent ") with help dissolution treatment agent with And the gathering of agitation induction is avoided with protection treatment protein, it also allows for preparaton when being exposed to shear surface stress not Cause active treatment protein or antibody degeneration.Nonionic surfactant with about 0.05mg/ml to about 1.0mg/ml, or The scope of about 0.07mg/ml to about 0.2mg/ml exists.In some embodiments, nonionic surfactant is with about The scope of 0.001% to about 0.1%w/v or about 0.01% to about 0.1%w/v or about 0.01% to about 0.025%w/v exists.

Suitably nonionic surfactant includes Polysorbate (20,40,60,65,80 etc.), Polyoxamer (184,188 etc.),Polyhydric alcohol,Polyoxyethylenesorbitans monoether (Deng), Lauromacrogol 400, Polyoxyl 40 stearate, polyoxyethylene Castor oil hydrogenated 10,50 and 60, glyceryl monostearate, sucrose fatty acid ester, methylcellulose and carboxymethyl cellulose.Can make Anionic detergent include sodium lauryl sulphate, dioctyl sodium sulphosuccinate and sodium cetanesulfonate.Cation Detergent includes benzalkonium chloride or benzethonium chloride.

In order to preparaton is used for internal, they must be aseptic.Filtered by sterilised membrane filter, so that preparation Agent becomes aseptic.Typically therapeutic composition herein is inserted in the container with sterile access port, such as, there is skin The intravenous solution bag of the stopper that hemostasis syringe needle is pierceable or phial.

Administration route, according to known and acceptable method, is such as injected by the one or many of suitable method or one Infusion in the long section time, such as, by subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, in pathological changes or intra-articular route Injection or infusion, surface applied, suck or by sustained release or extend release means.

Preparaton herein can also contain have more than a kind of, active ingredient necessary to treated specific adaptations disease Thing, preferably those complementary activities and not adversely affect each other.Or compositions can comprise cytotoxic agent, cell because of Son or growth inhibitor.This quasi-molecule suitably effectively to measure combination existence for predetermined purpose.

V. Therapeutic Method

Provided herein for treating in experimenter or preventing by the side of the cell-mediated disease expressing Siglec-8 Method, it includes using this experimenter the described herein anti-Siglec-8 antibody of effective dose, and (the anti-Siglec-8 of such as humanization resists Body) or a combination thereof thing.In some embodiments, this experimenter (such as people patient) has Eosinophils mediate the most after diagnosing Disease or the disease of risky generation Eosinophils mediate.In some embodiments, this experimenter (such as people patient) is There are disease or the disease of risky this mast cell mediated of generation of mast cell mediated after diagnosing.In some embodiments, This experimenter has the disease of Eosinophils mediate or the disease of mast cell mediated.

Abatement provided herein or reduce eosinophilic method, it this paper institute including experimenter uses effective dose State anti-Siglec-8 antibody (such as humanization anti-Siglec-8 antibody).In some embodiments, eosinophilic abatement Or minimizing is by comparing with this antibody treatment eosinocyte group in the sample (such as tissue sample) of experimenter later Number comes what the eosinocyte group's number in the sample of experimenter was measured with by this antibody treatment.In some embodiments In, eosinophilic abatement or minimizing are (such as to organize sample from the sample of experimenter later by this antibody treatment by comparing Product) in eosinocyte group's number with from the eosinocyte group in the sample of another experimenter of not this antibody treatment Number or from the sample of the experimenter of not this antibody treatment average eosinocyte group's number measure.Real at some Executing in scheme, this sample is tissue sample (such as lung sample, nasal polyp sample, etc.).In some embodiments, addicted to eosin The abatement of cell or minimizing are the eosinophilic apoptosis owing to having activated.Eosinocyte can be by cytokine or sharp Usually activation or sensitization, such as, but not limited to IL-5, GM-CSF, IL-33, IFN-γ, TNF-α, and leptin.Implement at some In scheme, eosinophilic abatement or minimizing are due to the eosinophilic apoptosis of tranquillization.In some embodiments, addicted to daybreak Erythrocytic abatement or minimizing are due to the cytotoxicity (ADCC) of antibody dependent cellular mediation.In some embodiments, Stop or reduce eosinocyte and generate inflammatory mediators.Exemplary inflammatory mediators includes but not limited to reactive species of oxygen, (eosinocyte derives corpuscular protein (granule protein) for such as Eosinophil Cationic Protein, major basic protein Neurotoxin, eosinophil peroxidase, etc.), lipid mediator (such as PAF, PGE1, PGE2, etc.), enzyme is (such as Elastoser), somatomedin (such as VEGF, PDGF, TGF-α, TGF-β, etc.), chemotactic factor (such as RANTES, MCP-1, MCP-3, MCP4, eosinophil activation chemotactic factor (eotaxin), etc.) and cytokine (such as IL-3, IL-5, IL-10, IL-13, IL-15, IL-33, TNF-α, etc.).

Abatement or the method that reduce mastocyte, it this paper institute that include experimenter use effective dose are also provided herein State anti-Siglec-8 antibody (such as humanization anti-Siglec-8 antibody).In some embodiments, the abatement of this mastocyte Or minimizing is by comparing by this antibody treatment later from the sample (such as tissue sample or biological fluid sample) of experimenter In mastocyte group's number come what mastocyte group's number in the sample of experimenter was measured with by this antibody treatment.? In some embodiments, the abatement of mastocyte or minimizing are by comparing by this antibody treatment later from the sample of experimenter Mastocyte group's number in (such as tissue sample or biological fluid sample) is subject to from another of not this antibody treatment Mastocyte group's number or thin from the average hypertrophy in the sample of the experimenter of not this antibody treatment in the sample of examination person Born of the same parents group's number is measured.In some embodiments, this sample is tissue sample (such as skin samples, lung sample, bone marrow sample Product, nasal polyp sample, etc.).In some embodiments, this sample is biological fluid sample (such as blood sample, a gas Pipe bronchoalveolar lavage fluid sample, and Nasal lavage fluid sample).In some embodiments, the abatement of mastocyte or minimizing be due to The cytotoxicity (ADCC) of antibody dependent cellular mediation.In some embodiments, abatement or the minimizing of mastocyte is to subtract Less or stop mastocyte to generate the preformed or inflammatory mediators that is newly formed.Exemplary inflammatory mediators includes but not limited to Histamine, N-.alpha.-Methylhistamine, enzyme (such as trypsinlike enzyme (tryptase), chymase (chymase), cathepsin G, Carboxypeptidase, etc.), lipid mediator (such as Prostaglandin D2, PGE2, leukotriene B4, leukotriene C, platelet activation The factor, 11-β-prostaglandin F2, etc.), ((i.e. eosinophil activation becomes chemotactic factor for such as CCL2, CCL3, CCL4, CCL11 Change the factor), CXCL1, CXCL2, CXCL3, CXCL10, etc.), and cytokine (such as IL-3, IL-4, IL-5, IL-15, IL- 33, GM-CSF, TNF, etc.).

Method that abatement express the mastocyte of Siglec-8 is also provided herein in experimenter, and it includes this tested Person uses the described herein anti-Siglec-8 antibody (such as humanization anti-Siglec-8 antibody) of effective dose, and wherein this resists Siglec-8 antibody kills the mastocyte expressing Siglec-8 by ADCC activity.In some embodiments, with process before Baseline values compare, this anti-Siglec-8 antibody is loose thin by the expression Siglec-8's in the sample that this experimenter obtains Born of the same parents' abatement at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100%.? In some embodiments, compared with baseline values before treatment, the sample that this anti-Siglec-8 antibody will obtain from this experimenter In expression Siglec-8 mastocyte abatement at least about 20%.In some embodiments, the abatement of mastocyte or kill Wound is by comparing by this antibody treatment later in the sample (such as tissue sample or biological fluid sample) of experimenter Mastocyte group's number comes what the mastocyte group's number in the sample of experimenter was measured with by this antibody treatment.At some In embodiment, the abatement of mastocyte or killing are by comparing by this antibody treatment later from the sample of experimenter (such as Tissue sample or biological fluid sample) in mastocyte group's number and another experimenter from not this antibody treatment Mastocyte group's number in sample or from the average mastocyte group's number in the sample of the experimenter of not this antibody treatment Range estimation amount.In some embodiments, this sample is tissue sample (such as skin samples, lung sample, bone marrow specimens, breath Disease of muscle sample, etc.).In some embodiments, this sample is biological fluid sample (such as blood sample, bronchovesicular Irrigating solution sample, and Nasal lavage fluid sample).In some embodiments, this anti-Siglec-8 antibody the most engineered with Improve ADCC activity.In some embodiments, this anti-Siglec-8 antibody comprise in Fc district at least one place improve ADCC live The amino acid replacement of property.In some embodiments, the heavy chain at least one of this antibody or two are non-fucosylations.? In some embodiments, the abatement of mastocyte or killing reduce or stop mastocyte generate preformed or be newly formed Inflammatory mediators.Exemplary inflammatory mediators includes but not limited to histamine, N-.alpha.-Methylhistamine, and (such as trypsinlike enzyme, class is rotten for enzyme Protease, cathepsin G, carboxypeptidase, etc.), lipid mediator (such as Prostaglandin D2, PGE2, leukotriene B4, Leukotriene C, platelet derived growth factor, 11-β-prostaglandin F2, etc.), chemotactic factor (such as CCL2, CCL3, CCL4, CCL11 (i.e. eosinophil activation chemotactic factor), CXCL1, CXCL2, CXCL3, CXCL10, etc.), and cytokine (such as IL-3, IL-4, IL-5, IL-13, IL-15, IL-33, GM-CSF, TNF, etc.).

The method that the activity of suppression mast cell mediated is also provided herein, it basis including experimenter uses effective dose The described anti-Siglec-8 antibody of literary composition (such as humanization anti-Siglec-8 antibody).In some embodiments, mast cell mediated Activity suppression be by comparing by this antibody treatment later from the sample (such as tissue sample or blood sample) of experimenter In the activity of activity and the mast cell mediated come in the sample of experimenter by this antibody treatment of mast cell mediated Measure.In some embodiments, the suppression of the activity of mast cell mediated is by comparing by this antibody treatment later certainly The activity of the mast cell mediated in the sample (such as tissue sample or biological sample) of experimenter with from not this antibody The activity of the mast cell mediated in the sample of another experimenter processed or the sample of the experimenter from not this antibody treatment The activity measurement of the average mast cell mediated in product.In some embodiments, this sample is tissue sample (such as skin Sample, lung sample, bone marrow specimens, nasal polyp sample, etc.).In some embodiments, this sample is biological fluid sample (such as blood sample, bronchoalveolar lavage fluid sample, and Nasal lavage fluid sample).In some embodiments, mastocyte The suppression of the activity of mediation is the suppression of mastocyte threshing.In some embodiments, active the pressing down of mast cell mediated System is the suppression of airway smooth muscle contraction.In some embodiments, the suppression of the activity of mast cell mediated is mastocyte In the suppression of calcium current.In some embodiments, the suppression of the activity of mast cell mediated is that mastocyte release is pre-formed Or the suppression of inflammatory mediators that is newly formed.Exemplary inflammatory mediators includes but not limited to histamine, N-.alpha.-Methylhistamine, enzyme (such as trypsinlike enzyme, chymase, cathepsin G, carboxypeptidase, etc.), lipid mediator (such as Prostaglandin D2, PGE2, leukotriene B4, leukotriene C, platelet derived growth factor, 11-β-prostaglandin F2, etc.), chemotactic factor is (such as CCL2, CCL3, CCL4, CCL11 (i.e. eosinophil activation chemotactic factor), CXCL1, CXCL2, CXCL3, CXCL10, etc.), With cytokine (such as IL-3, IL-4, IL-5, IL-13, IL-15, IL-33, GM-CSF, TNF, etc.).

For prevention or the treatment of disease, the optimal dose of activating agent can depend on disease to be treated type (as Defined above), the order of severity of disease and process, use medicament and be in order at prevention purpose or therapeutic purposes, previous Therapy, the clinical history of experimenter and the response to medicament, and the judgement of attending doctor.Described medicament is suitable for The treatment of row is applied to experimenter.In some embodiments of methods described herein, described anti-Siglec-8 antibody is used Between interval be about one month or longer.In some embodiments, the interval between using is about two months, about three Month, about four months, about five months, about six months or longer.As used in this article, use between interval refer to applied once resist Time period between body and next time administration of antibodies.As used in this article, the interval of about month includes surrounding.Thus, In some embodiments, the interval between using is about 4 weeks, about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 24 weeks, or more Long.In some embodiments, described treatment includes repeatedly using described antibody, and the interval between wherein using can change. Such as, the interval between using for the first time and using for the second time is about one month, and the interval between subsequent applications is about three Month.In some embodiments, use for the first time and second time use between interval be about one month, second time is used and the Interval between using for three times is about two months, and the interval between subsequent applications is about three months.In some embodiments, Anti-Siglec-8 antibody described herein is used with flat dosage (flat dose).In some embodiments, with about 150 to about Experimenter is used anti-Siglec-8 antibody described herein by the dosage of 450mg every dose.In some embodiments, with about 150mg, Experimenter is used described anti-Siglec-8 by dosage arbitrary for 200mg, 250mg, 300mg, 350mg, 400mg, and 450mg every dose Antibody.In some embodiments, the dosage with about 0.1mg/kg to about 10mg/kg or about 1.0mg/kg to about 10mg/kg will Anti-Siglec-8 antibody described herein is applied to experimenter.In some embodiments, with about 0.1mg/kg, 0.5mg/kg, 1.0mg/kg, 1.5mg/kg, 2.0mg/kg, 2.5mg/kg, 3.0mg/kg, 3.5mg/kg, 4.0mg/kg, 4.5mg/kg, 5.0mg/kg, 5.5mg/kg, 6.0mg/kg, 6.5mg/kg, 7.0mg/kg, 7.5mg/kg, 8.0mg/kg, 8.5mg/kg, Anti-Siglec-8 antibody described herein is applied to experimenter by the arbitrary dosage of 9.0mg/kg, 9.5mg/kg, or 10.0mg/kg. Any dosed administration frequency mentioned above can be used.

A kind of Therapeutic Method being contemplated herein is with anti-Siglec-8 Antybody therapy Eosinophils mediate described herein Disease and/or the disease of mast cell mediated.The disease of Eosinophils mediate includes migrating with eosinocyte, chemotactic Property, generate, or be granulated relevant disease or disease.Similarly, the disease of mast cell mediated includes and mast cell migration, becomes The property changed, generates, or is granulated relevant disease or disease.Disease with the medicable Eosinophils mediate of the preparaton of the present invention And/or the disease of mast cell mediated includes asthma, allergic rhinitis, nasal polyp, atopic dermatitis, chronic urticaria (example Such as chronic idiopathic urticaria and Chronic Spontaneous urticaria), Mastocytosis, eosinophilic leukemia, and addicted to daybreak Erythrocytosis syndrome.Disease and/or mastocyte with the medicable Eosinophils mediate of the preparaton of the present invention are situated between The disease led also includes few granulocytic asthma, acute or chronic airways hypersensitivity, eosinophilic esophagitis, Qiu-execute Two Cotards, the inflammation relevant with cytokine, the inflammation relevant with the cell expressing Siglec-8, with expression Relevant pernicious of the cell of Siglec-8, physical urticaria, cold urticaria, pressure urticaria, epidermolysis class sky bleb Skin ulcer, food anaphylaxis, and allergic bronchopulmonary aspergillosis (ABPA).

In some embodiments of method in this article, anti-Siglec-8 antibody suppression allergia provided herein is anti- One or more symptoms answered.In some embodiments, this atopic reaction is I type hypersensitivity reaction.

Allergic rhinitis (also referred to as allergic rhinoconjunctivitis or hay fever) are the atopic reactions to the allergen sucked Most common performance, its order of severity is usually relevant with the intensity of the exposure to allergen and time span with the persistent period.It It is a kind of chronic disease, can come across any age first, but the childhood period of being that typically in or juvenile era outbreak.Typical onset includes A large amount of watery rhinorrheas, paroxysmal sneeze, nasal obstruction is, and the scratching where it itches of nose and palate.After nose, mucus drainage also causes throat pain, hawk and Cough.Can also there is the symptom of the allergia blepharoconjunctivitis, with strongly scratching where it itches of conjunctiva and eyelid, rubescent, shed tears, and fear Light.Serious attack is frequently accompanied by general malaise, weak, tired, and sometimes, the muscular soreness of strong sneeze after date.

Asthma (also referred to as reversible obstructive airway disease) is characterised by that tracheobronchial tree is to respiratory irritation and a gas The hyperresponsiveness of pipe contractility chemical drugs, produce wheezing, dyspnea, feeling of chest tightness, and cough outbreak, they be from That send out reversible or reversible by treatment.It is a kind of chronic disease relating to whole air flue, but the order of severity is from accidental light Temporarily outbreak is to severe for degree, for a long time, life-threatening bronchial obstruction and change.The sign of asthma attack includes rapid breathing, Audible wheezing, and use accessory respiratory muscle.There is typically further rapid pulse and blood pressure raises, rise in nose secretion and peripheral blood High levels of eosinophils.Asthma and atopy can coexist, but the most approximately half of asthma patient is also atopic, And the atopy patient of the least percentage ratio also has asthma.But, atopy and asthma are not completely self-contained, because of For compared with ergotropy individuality, asthma more frequently being occurs in atopic individuals, especially childhood period.Asthma is in group Knit and on, be divided into two subgroups, i.e. extrinsic asthma and intrinsic asthma further.It addition, asthma involves the chronic of air flue Inflammation, the acute exacerbation that between different patients, frequency difference and response environment trigger thing and occur.In several cases, gas can occur The chronic of road is reinvented.

Extrinsic asthma (also referred to as allergia, atopy or immunology asthma) describes typically at life in early days, generally exists Infancy stage or childhood period occur asthma patient.Other performance (including eczema or allergic rhinitis) atopic usually coexists. Asthma attack can exist in the case of animal during pollen season, or is being exposed to room dirt, and feather is rested the head on, or other allergen Shi Fasheng.Skin test shows the positive welt to pathogenic allergen and flare reaction.It is interesting that serum total Ig E concentration may often be such that Raise, but be normal sometimes.Intrinsic asthma (also referred to as anallergic or essential asthma) generally betides first Adult age, after apparent respiratory tract infection.Symptom includes and pollen season or be exposed to unrelated chronic of other allergen or multiple The property sent out bronchial obstruction.Skin test to common atopen is negative, and serum IgE concentration is normal.Other symptom Including expectorant blood and eosinophilia.For the purpose of present patent application, term " disease of Eosinophils mediate " includes becoming Answering property and anallergic both asthma.In some embodiments, disease and/or the hypertrophy with Eosinophils mediate are thin The experimenter of the disease of born of the same parents' mediation suffers from by suction-type corticosteroid, fugitive β2agonists, long-acting β2agonists, or its group Close the asthma the most appropriately controlled.

Atopic dermatitis (also referred to as eczema, neurodermatitis, atopic eczema or Besnier (Besnier) prurigo) is The common chronic skin disease special to a patient subgroups, this patient subgroups has the spy of familial and amynologic characteristic should Property.Substitutive characteristics is pruritus scytitis response, its induced character, and the skin macule of symmetry distribution, to some portion There is preference position.Although atopic dermatitis is classified as the atopy of cutaneous form (because it is with allergic rhinitis with asthma and high IgE Level is relevant), but the order of severity of dermatitis not always relevant with allergen exposure during skin test, and desensitize and (become with other Answering property disease is different) it not effective Therapeutic Method.Although high SERUM IgE can confirm the diagnosis of allergic asthma, but normal water The flat diagnosis that can not get rid of allergic asthma.The outbreak of this disease can betide any age, and pathological changes starts urgency, has red Speckle edema pimple or the speckle with decortication.Scratch where it itches and cause sepage and incrustation, the chronicest lichenification.At cellular level, Acute pathological changes is hydropigenous, and corium is impregnated with mononuclearcell, i.e. CD4 lymphocyte.Neutrophil(e) cell, thin addicted to eosin Born of the same parents, plasma cell and basophil are rare, but there is the mastocyte of threshing.The feature of chronic disease is that epidermis increases Raw, hyperkeratosis and parakeratosis, and also corium is impregnated with mononuclearcell, Lang Gehansishi (Langerhans) cell and fertilizer Maxicell.Can also there is Fibrotic focal zone, including the perinenrial participation of nervelet.

Urticaria and angioedema refer to health swelling, erythema and scratch where it itches that (it is derived from shallow table skin heart and is stung by histamine The receptor swashed), and be the hallmark cutaneous feature of general anaphylaxis reaction.General anaphylaxis reaction is derived from medicine, insecticide Venom or food, the reaction of simultaneous IgE mediation in multiple organ.It is to be induced by allergen, and mastocyte loads IgE and cause suddenly, cause deep and life-threatening, the change of the function of multiple vital organ.Blood vessel subsides, acute Airway obstruction, cutaneous vasodilation and edema, and gastrointestinal and urogenital muscle spasm occur the most simultaneously, although and not always Same degree.Anaphylactoid pathology include angioedema and lung hyperinflation, with Air way mucus filling with focal Pulmonary atelectasis.At cellular level, the performance of lung is similar with the acute asthmatic attack phase, too much with bronchial submucosa glandular secretion, Mucosa and submucosa showed edema, thin addicted to eosin in peribronchial blood vessel congestion/hyperemia (congestion) and bronchial wall Born of the same parents increase.Pulmonary edema and hemorrhage can be there is.Bronchial muscle spasm, hyperinflation, and even rupture of alveoli can also be there is.People Anaphylactoid principal character includes edema, in blood vessel congestion/hyperemia, and the lamina propria of larynx, trachea, epiglottis and hypopharynx addicted to daybreak Erythrocytosis.Exposure to allergen can be injected via ingesting, suction or skin or mucosal contact.Reaction is being exposed to Start in several seconds or several points after allergen.Can there is approaching initially the fearing or sensation, followed by one or more targets of misfortune Tract: cardiovascular, breathes, the symptom in skin or gastrointestinal.Allergen responsible to anaphylaxis is different from those The allergen the most relevant with atopy.Food, medicine, insecticide venom or latex are frequent origins.Food allergens includes that A bit crustacean, Mollusca (such as Lobster, shrimp, Eriocheir sinensis), fish, bean (such as Semen arachidis hypogaeae, Semen Pisi sativi, Kidney bean (bean), Radix Glycyrrhizae), plant Son (such as Semen Sesami, Semen Gossypii, caraway (caraway), Semen Sinapis, Semen Lini, Helianthi), nut, berry, albumen, Fagopyrum esculentum Moench powder and Ruzhong is found.Medicine allergen includes what those found in heterologous protein and polypeptide, polysaccharide and hapten medicine.Insecticide Allergen includes hymenopteran, including Apis, yellow jacket (yellow jacket), hornet (hornet), wasp (wasp) With fire ant.Although epinephrine is anaphylactoid typical treatment, but hydryllin or other histamine blockers are generally at place For alleviating serious urticaria or angioedema reaction in side.

Nasal polyp is a kind of chronic inflammatory disease of upper respiratory tract, is characterised by that Inflamed tissue grows entrance nasal cavity, and Although and still do not know the definite cause of disease, but known there is 1 to 5% adult popularity (Settipane G A: Epidemiology of nasal polyps.Allergy Asthma Proc,1996,17:231-236).Nasal polyp leads to It is normally present in 20 years old or in more old male, and causes nasal obstruction, hyposmia, and send out infection repeatly, have than allergia all the year round (Li et al., the Characterizing T-Cell Phenotypes in of the impact on quality of life that rhinitis is the biggest Nasal Polyposis in Chinese Patients,J Investig Allergol Clin Immunol,2009； Vol.19(4):276-282).All nasal polyp patients of up to 1/3rd report have asthma, but the heavy breathing of only 7% Breathe heavily patient and there is nasal polyp.The major cell types involving nasal polyp includes eosinocyte and mastocyte.

VI. goods or test kit

On the other hand, it is provided that comprise goods or the test kit of anti-Siglec-8 antibody described herein.Described goods or reagent Box can further include about the description using described antibody in the method for the invention.As follows, in certain embodiments, Described goods or test kit comprise about at disease and/or the hypertrophy for treating or prevent Eosinophils mediate in individuality Using the description of anti-Siglec-8 antibody in the method for cell-mediated disease, the method includes individuality is used effective dose Anti-Siglec-8 antibody.In certain embodiments, described individuality is people.In some embodiments, described individuality has choosing Disease from lower group: asthma, allergic rhinitis, nasal polyp, atopic dermatitis, chronic urticaria, Mastocytosis, addicted to daybreak Fragility of erythrocytes leukemia, and eosinophilia's syndrome.In certain embodiments, described individuality has selected from lower group Disease: few granulocytic asthma, acute or chronic airways hypersensitivity, eosinophilic esophagitis, Qiu-execute Er Shi is comprehensive Levy, the inflammation relevant with cytokine, the inflammation relevant with the cell expressing Siglec-8, with the cell expressing Siglec-8 Relevant is pernicious, physical urticaria, cold urticaria, pressure urticaria, bullous pemphigoid, food anaphylaxis, and Allergic bronchopulmonary aspergillosis (ABPA).

Described goods or test kit can further include container.Suitably container includes such as bottle, phial (such as dual chamber Phial), syringe (such as single chamber or double-chamber syringe) and test tube.Described container can be with various materials such as glass or mould Material is made.This container is equipped with preparaton.

Described goods or test kit can further include label or package insert, its on container or with described container phase Even, can indicate whether the explanation rebuild about preparaton and/or use.Described label or package insert can indicate described preparation further Agent can be used for or be intended for subcutaneous, intravenous, or the using to treat in individuality or prevent eosinocyte of other pattern The disease of mediation and/or the disease of mast cell mediated.The described container equipped with preparaton can be the phial being intended for single use Or nonexpondable phial, it allows the repetitive administration rebuilding preparaton.Described goods or test kit can further include Two containers, wherein equipped with suitable diluent.Described goods or test kit can farther include from business, treatment, and user Position sees other material wanted, and including other buffer agent, diluent, filter, syringe needle, syringe, and is printed on operation instructions Package insert.

In a specific embodiment, the present invention is provided to single dose and use the test kit of unit.This type of test kit Comprise the container of the aqueous preparaton of therapeutic antibodies, including single chamber or multicell pre-filled syringe.Exemplary prefilled injection Device can obtain from Vetter GmbH (Ravensburg, Germany).

Goods or test kit herein comprise the container equipped with the second medicine the most further, wherein said anti- Siglec-8 antibody is the first medicine, and these goods or test kit comprise further about with effectively on label or package insert Measure by the description of described second Drug therapy experimenter.Described the second exemplary medicine can be anti-IgE antibodies, antihistamine Agent, bronchodilator, glucocorticoid, NSAID, decongestant, anti-tussive agents, analgesic, TNF-antagonist, integrin is short of money Anti-agent, immunosuppressant, IL-4 antagonist, IL-13 antagonist, dual IL-4/IL-13 antagonist, DMARD, with B cell surface The antibody that mark combines, and/or BAFF antagonist.

In another embodiment, goods provided herein or test kit, it comprises preparaton described herein, It is for using in autoinjector device.Autoinjector can be described as be at after starting without other from patient or execute The required action of user will deliver the injection device of its content.When delivering, speed is necessary constant and time of delivery is more than a little For a moment the when of (a few moment), they are particularly suitable for the self-medication of therapeutic preparaton.

By with reference to following embodiment, the present invention can be more fully appreciated with.But, they should not be construed as and limit this Bright scope.Be appreciated that embodiment described herein and embodiment the most for exemplary purposes, and according to they, this area Technical staff will recognize that various change and change, and they are included in spirit and scope and claims In the range of.

Embodiment

Siglec (bound sialic acid, immunoglobulin-like agglutinant) is the single pass transmembrane mainly found on the leukocytes Cell surface protein.One member of Siglec family, Siglec-8 is initially as identifying novel people's eosinocyte albumen A part for the achievement of matter finds.Beyond being expressed by people's eosinocyte, it is also by mast cell-expressed.Siglec-8 knows Not a kind of sulfated glycan, 6 '-sulfo group-saliva acyl group Lewis X, and press down containing intracellular immunity receptor is based on tyrosine Property motif (ITIM) domain processed, it demonstrates suppression mastocyte function.For the murine antibody of Siglec-8,2E2 and 2C4 resists Body is described in United States Patent (USP) No.8,207,305；United States Patent (USP) No.8,197,811, United States Patent (USP) No.7,871,612, and the U.S. Patent No.7,557,191.The heavy chain variable domain of little mouse-anti Siglec-8 2C4 antibody and the aminoacid sequence of light variable domains Row can be shown in such as United States Patent (USP) No.8,207,305, and it is SEQ ID NO:2 and SEQ ID NO:4 respectively.

Embodiment 1: the generation of inosculating antibody Siglec-8 antibody and sign.

From mice 2E2 antibody and mice 2C4 antibody tormation chimeric antibody, and with post analysis, the combination of people Siglec-8 is lived Property.

Method and result

Chimeric 2E2 antibody (ch2E2) and the generation of chimeric 2C4 antibody (ch2C4)

In order to generate chimeric 2E2 antibody (ch2E2), use RNeasy Mini test kit (Qiagen) according to manufacturer Scheme processing freezes Mouse Hybridoma Cells 2E2 cell lysate suddenly to separate total serum IgE.Use the first chain cDNA synthetic agent box (GE Life Sciences) according to manufacturer scheme reverse transcription 3 μ g separate RNA sample to generate 2E2cDNA.Use subsequently The big divisional mixture of Phusion Flash High fidelity PCR (Thermo Scientific) expands 2E2cDNA by PCR, and really Recognize the sequence in PCR reaction.The big divisional mixture PCR of Phusion High fidelity PCR is used to expand immunoglobulin heavy chain variable region (VH) cDNA and Kappa variable region of light chain (VL).The result of each PCR reaction is all single amplified production, uses QIAquick PCR purification kit (Qiagen) is according to the scheme purification of manufacturer, and obtains the sequence of every immunoglobulin chain.Kappa The consensus sequence of variable region of light chain is referred to as 2E2VK, and the consensus sequence of heavy and light chain variable region is referred to as 2E2VH.2E2VK protein sequence Arrange identical with the protein sequence of mice 2C4IgG1 antibody (see Kikly et al., J.Allergy Clin Immunol., 2000；105:1093-1100), except first residue, Gln Glu replaces there, and 2E2VH protein sequence and 2C4 Protein sequence identical.

The structure of chimeric viral vectors needs to use ligase not dependent clone that the variable region clone of amplification is entered IgG/ Kappa constant region carrier.In short, digest pCMV carrier with BfuA1 (BsPM1), by the gel purified load through digestion Body, and generate the compatible pendency in carrier by incubation together with T4DNA polymerase and 100mM dATP.For insert, use The beginning (i.e. reverse primer) of the 3 ' ends (i.e. forward primer) or constant region (IgG1 or Kappa) containing targeting sequencing, being followed by can The primer of the beginning (in each direction) becoming district expands antibody sequence by PCR from 2E2cDNA.Use PCR purification kit (Qiagen) insert of purification amplification, and by the life in insert of incubation together with T4DNA polymerase and 100mM dTTP Become complementation pendency.In incubation at room temperature carrier and insert, and it is used for converting Competent TOP10 antibacterial (Invitrogen), Coated plate on the culture plate containing kanamycin subsequently.Separate several clone, and screen bacterium colony by PCR.Select containing correct The clone of the PCR primer of size, uses Miniprep Kit (Qiagen) to separate DNA, and to DNA sequencing.

In order to generate chimeric 2C4 antibody (ch2C4), synthesis (GeneScript) 2C4 variable region of heavy chain (VH) and Kappa are light Chain variable region (VK), and apply the same procedure for cloning chimeric 2E2 antibody.Use and Expi293 expression system test kit (Invitrogen) ExpiFectamine 293 reagent provided together according to manufacturer scheme encode ch2E2 heavy chain and The construction of ch2E2 Kappa light chain or coding ch2C4 heavy chain and construction (each 1 μ g DNA) cotransfection of ch2C4 Kappa light chain The Expi293 suspension cell (HEKC) of growth in Expi293 transfection media (Invitrogen) and antibiotic.Will Cell is cultivated 7 days in 2ml growth medium in 6 orifice plates, gathers in the crops culture medium afterwards, and measures recombiant protein by ELISA Express.

The Siglec-8 of chimeric antibody combines activity

Chimeric 2E2 and the chimeric 2C4IgG1K antibody knot to recombined human Siglec-8 extracellular domain (ECD) is measured by ELISA Close.For Siglec-8 binding assay, the following 384 hole SpectraPlate (Perkin Elmer) that prepare, i.e. every with 30 μ L Hole 0.4 μ g/mL Siglec-8ECD is coated overnight in 4 DEG C, then passes through and cleans in cleaning buffer solution (0.1%Tween 20) Hole is removed and is coated solution, and closes 2 hours in room temperature with 90 μ L 5%BSA/TBS solution.By chimeric antibody (ch2E2 or Ch2C4) or the mouse antibodies (m2E2 or m2C4) 3 times of serial dilutions in 0.2%BSA/TBS (initial concentration is 5000ng/ ML) add to each through coated hole.By plate in incubation at room temperature 2 hours, clean-out opening subsequently, it is added on 0.2%BSA/ afterwards In TBS solution, Goat anti human's Fc peroxidase conjugated thing (1:10000 dilution) or the anti-mouse Fc peroxidase of dilution are sewed Compound (1:30000 dilution).By plate in incubation at room temperature 45 minutes, then clean, and each hole is added 30 μ L K-blue HRP Substrate (SkyBio Ltd).After incubation at room temperature 15 minutes, stop solution (SkyBio by each hole being added 10 μ L Red Ltd) stopped reaction is carried out.ELISA readout instrument PHERAStar FS (BMG Labtech) is used to read optical density or reality in 650nm Test sample.

Two kinds of chimeric antibodys are with suitable EC₅₀Value combines complete S iglec-8ECD.Chimeric antibody ch2E2 with little mouse-anti Body m2E2 compares lower EC₅₀Value combines Siglec-8ECD (table 2).

Table 2.The combination of antibody on human Siglec-8ECD.

	m2E2	Ch2E2 purification	ch2E2	ch2C4
					EC50	0.1003	0.05701	0.04759	0.07411

Embodiment 2: the generation of humanization anti-Siglec-8 antibody and sign.

The sequence using chimeric antibody 2E2 and chimeric antibody 2C4 designs the humanization pattern of mice 2E2 antibody.

Method and result

The design of humanization anti-Siglec-8 antibody

Use from International Immunogenetics Database 2009 (Lefranc, MP., Nucleic Acid Res., 2003,31 (1): 307-10) and Kabat Database Release 5of Sequences of Proteins of Immunological Interest (last renewal on November 17th, 1999) (Kabat et al., NIH National Technical Information Service, 1991,1-3242) people and mouse immuning ball protein Protein sequence collects the data base of human normal immunoglobulin's sequence in Kabat comparison.The data base of compilation contains 10,906 kinds VH sequence and 2,912 kinds of VK sequences.

Use the Modeller program (Eswar that Discovery Studio bag (Accelrys, Inc.) includes Et al., Curr.Protoc.Bioinformatics, 2006, Ch.5:Unit 5.6) calculate mouse antibodies 2C4 variable region Homology model.The atomic coordinates of 1a7O.pdb, 1dqd.pdb and 1ORS.pdb be respectively VH, VL and main chain/interface High framework homogeneity sequence template, such as the basic Local Alignment Search by antibody pdb structural database (Accelrys, Inc.) Mensuration analyzed by instrument (BLAST).Using these templates is that framework generates 30 kinds of initial models, and uses minimum energy model raw Become 20 kinds of ring models (including all CDR rings), by its 5 kinds of optimal ring templates, use Kabat definition to ultimately produce final Mice 2C4 model.

Humanization needs to identify suitable people V district.Gibbs sequence analysis programs (MRCT) is used to use various selection standard With 2C4VH and VK protein sequence queries people VH and VK data base.Use Discovery Studio program (Accelrys), mirror Determine the final Homology model middle-range CDR residue (Kabat definition) of mouse antibodies 2C4Interior framework (FW) residue, and be referred to as “CDR involucrum ".Nobody's VH sequence with have heightThe 2C4 of CDR involucrum share CDR 1,2 and 3 length homogeneity and/or VCI homogeneity is shared with 2C4VH.AF471521 is next optimal candidate, and CDR3 size is 14 residues, crucial Framework residues (19/24Involucrum and 18/22VCI) there is highest identity score, and other sequence has other difference so that they It is in lower priority.But, AF471521 has somatic mutation at 11 from its germline VH gene X92218.In order to subtract Light somatic mutation, by any be different from germline and/or in mice unreserved Framework residues back mutation human germline's sequence Row.Therefore, 6 Framework residues back mutations are become germline, and remain 5 residues being different from germline be crucial Framework residues and Identical with mouse sequence.

Since the Homology model having used 2C4 antibody identifies a kind of suitably receptor people's framework, then by inciting somebody to action The CDR of 2E2 antibody is grafted onto protein and the DNA sequence designing synthesis on this receptor people's framework.The initial designs of 2E2RHA CDR 1,2 and 3 grafting from 2E2VH is entered in the receptor FW of AF471521.2E2VH CDR (gray shade) enters The insertion of AF471521FW sequence produces initial humanization variant, the design (Fig. 1) of 2E2RHA.2E2RHA does not retain At Kabat position 24,48,49,67 and 68 5CDR involucrum/VCI residue, and will in humanization pattern 2E2RHB These back mutations become mice equivalent ones, or once suddenly change in following variant one: assemble sequence in a computer, and Named 2E2RHA to 2E2RHG (Fig. 1).

For humanization light chain, during similar to heavy chain, identify people's Kappa chain.AY867246 beCDR Involucrum/VCI aspect and 2E2VK have the sequence of highest identity but have somatic mutation at 6.Abandon by X93721 AY867246, X93721 contain 21/25Involucrum and 15/17 VCI and only 1 are from closest to germaine VK gene, X01668 Somatic mutation.Use DNA and the protein of the Frame Design humanization construction from X93721.By from 2E2VK's CDR 1,2 and 3 (gray shade) grafting enters in the receptor FW of X93721 to generate 2E2RKA (Fig. 2).2E2RKA does not has 4 not JoinCDR involucrum residue (i.e. 3,47,58 and 71), in variant 2E2RKB, back mutation becomes to be equal to mouse residues, or Sudden change individually in following variant: assemble sequence, and named 2E2RKA to 2E2RKG (Fig. 2) in a computer.

The generation of humanization anti-Siglec-8 antibody

The gene of synthesis (GenScript) 2E2RHA and 2E2RKA, and be people's sequence optimisation codon.Group in a computer Dress natural human Frame sequence AF471521 and X93721 (being weight and light chain respectively), and native mouse CDR sequence, and named 2E2RHA to 2E2RHG and 2E2RKA to 2E2RKG.Use software algorithm (GenScript), by silent mutagenesis optimization with Make the codon that employment cell preferentially utilizes, and synthesize.As expanded described in chimeric antibody about PCR the most in embodiment 1, RHA/B and RKA/B construction is expanded with primer PCR specific to expression vector and insert, not dependent by ligase Cloning reaction inserts in IgG/ Kappa constant region carrier, and if embodiment 1 is about described in generation chimeric antibody, is used for converting TOP10 antibacterial.Use QuickChange Lightning site directed mutagenesis kit (Stratagene) according to manufacturer subsequently Scheme modifies RKA and RHA to obtain everyone antibody variants (Fig. 1, Fig. 2, table 3, table 4, and table 5) by PCR mutation.

Table 3: the aminoacid sequence of the chimeric variable region with humanized antibody variants

Table 4: from 2E2 and the aminoacid sequence of the HVR of humanized antibody variants

Table 5: from 2E2 and the aminoacid sequence of the FR of humanized antibody variants

To cloning and sequencing, and plasmid miniprep kit (Qiagen) or PureYield plasmid is used to prepare system in a large number System (Promega) prepares expression plasmid DNA according to the scheme of manufacturer.As described in the most in embodiment 1, use encoding human Source or be fitted together to VH and VK expression plasmid prepared product transfection Expi293 cell (Invitrogen).In serum-free medium Cell is cultivated 7 days, hereafter from cell harvesting conditioned culture media, and measures to confirm the generation of antibody by ELISA.

The Siglec-8 of the antibody encoded by humanization VH and VK construction combines

By basic humanization weight and light chain and the pairing of their chimeric homologue, it is intended to identify humanization design any always Body problem.As described in Example 1, Siglec-8 antigen is used to measure antibodies by ELISA.2E2RHB is with chimeric Show more stronger than RHA heavy chain during light chain pairing, and RKB construction united with heavy chain chimeric construct thing (cVH) with than Both cVK constructions of pairing want high effect to combine Siglec-8 antigen.These results verifications weight and the people of both light chains Sourceization design the most correctly and all combines Siglec-8, however it is necessary that further work identifies that other having more preferably combines The humanized antibody of feature.

The Siglec-8 of the full-length human antibody combined with chimeric homologue combines

Combine full-length human antibody weight and light chain and compare to determine replacement with chimeric antibodyAnd canonical framework residue Humanization 2E2 whether it is enough to successfully with interface residue.With the different different humanization heavy chain vector of humanization light chain vector associating Combination cotransfection Expi293 cell.As described in Example 1, use Siglec-8 antigen to measure antibody by ELISA to tie Close.Compared with chimeric comparison, to the combination of Siglec8, these antibody show that HBKA and HBKB performance is more preferable than HAKA and HAKB Combination (table 6).

The Siglec-8 of full-length human antibody combines

In order to determine the relative importance that in heavy chain, single amino acid substitutes, with all light chain variant combinational expression humanizations Heavy chain RHC (A24V), RHD (V48L), RHE (S49G), RHF (F67L) and RHG (T68S), and with RHA and RHB pattern and chimeric Antibody control compares.The result of these antibodies Siglec-8ECD points out all these humanized antibodies to combine with identical Effect combines Siglec-8, except family RHA, RHF and RHG (and all light chain variant) (table 6).

Next determine to substitute with single amino acid from RKA, light chain is become whether RKB can affect antibodies.Assess by The knot of the antibody of the combination composition of all heavy chain mutant and light chain RKC (V3I), RKD (V48L), RKE (L47W) and RKF (E58V) Close, compare with chimeric antibody and RKA and RKB pattern.Do not show appreciable impact and combine the light chain variant (table 6) of pattern.Separately Outward, also checked the dependency of light chain germ line residue F71Y (RKG) in the combination of all heavy chain mutant, result proves this Residue generally causes combination to be substantially reduced.

Apparently, HEKA and HEKF is the best candidate antibodies for Siglec-8.Therefore, implement ELISA again to survey Try the antibody with efficient conjugated antigen, compare with chimeric antibody with as both HEKA and HEKF compareed.Result indicates Except the various combination of humanized heavy chain and humanization light chain combines Siglec-8, with the combination of RHA in a similar manner (table 6). It is good candidate that result has highlighted HEKA and HEKF, is comparatively speaking better than chimeric positive control and has irreducible minimum in the frame Degree mouse residues.

The generation of 2E2RH pattern 2 and CDR mutation variants

It is subsequently based on immediate germ line genes and generates a kind of humanized heavy chain.Other construction phase described above Same mode, uses the germline most like with 2E2VH, and IGHV4-59 Germline sequences (Fig. 1) designs the grafting of mice CDR, Synthesis (GenScript), and prepare, to generate humanized antibody, compare with the first pattern of RHA and RHB chain.It addition, be Whether mensuration antibody is resistant to some CDR3 residue becomes germline, is drawn in the CDR3 of RHE variant heavy chain by direct mutagenesis Enter and at 3, suddenly change (single and triple mutant) and introduce in the CDR3 of RKA and RKF light chain sudden change (Fig. 3) at 1.Use and retouch above The same procedure stated, expresses containing RHE sudden change or the antibody of RKA/RKF sudden change, and combines with whole group of complementary strand.

Heavy chain CDR grafting entered in people's germline compares with other heavy chain pattern.Directly grafting (RHA2) destroys completely Combination to antigen, andIt is non-with the first RHB variant that/VCI residue back mutation becomes the germline framework (RHB2) of mice to manifest The most similar, but containing 10 mouse residues compared with the 5 of RHB.Introduce prominent in the CDR3 of two chains in conjunction with data illustration The impact become, and point out the sudden change in heavy chain to have adverse effect to combining Siglec-8, and light chain mutant itself does not carries Improve, although best antibody is containing heavy chain candidate RHB (all mice back mutations) and RHE (table 6) for too many.

Table 6: the combination of antibody on human Siglec-8ECD.

Note: every hurdle represents an experiment；NA indicates non-availability.

The humanization candidate antibodies heat stability to high temperature

The relatively heat stability of humanized antibody.Antibody is made to stand the higher temperatures 10 minutes from-20 to 85 DEG C of changes, cold But to room temperature, and assess in ELISA algoscopy with the EC80 concentration of every kind of candidate.Antibody candidate is guided to show as stable (Fig. 4).It is fully inactive at 68 DEG C that HEKA/KF has the antibody that CDRL3 (i.e. the CDR3 of light chain) suddenlys change, and is entrenched in 70 DEG C of nothings Activity, guides candidate HEKA and HEKF still to have 25-50% activity in this temperature, the most fully inactive 75 DEG C of displays.

The mensuration of humanization candidate antibodies Tm

In order to measure the melting temperature guiding antibody, in two step affinity chromatographs and gel filtration system, purification is fitted together to, HEKA, HEKF with there is the identical humanization candidate that CDRL3 (i.e. the CDR3 of light chain) suddenlys change, and in hot transfer assay method survey Examination.The antibody of two kinds of variable concentrations incubation 71 together with fluorescent dye (Sypro Orange) is followed by qPCR thermal cycler Ring, each circulation raises 1 DEG C.Tm is defined as temperature during maximum fluorescence 50%.Chimeric and 5 kinds of humanized antibodies Tm confirm The result obtained in thermal stability determination method: most stable of antibody is HEKA and HEKF, they have other people than test Source antibody wants high Tm.HEKA has the Tm (Fig. 5 and Biao 7) higher than chimeric antibody.

Table 7: the chimeric and Tm of humanized antibody

Antibody	TM
		chVHVK 2uM	71℃
chVHVK 1uM	71℃
		HEKA 2uM	72℃
HEKA 1uM	72℃
		HEKF 2uM	70℃
HEKF 1uM	70℃
		HEKAmut 2uM	68℃
HEKAmut 1uM	68℃
		HEKFmut 2uM	67℃
HEKFmut 1uM	68℃

The affinity of humanization candidate antibodies and affinity

Use Biacore T200 to be analyzed by SPR and carry out Antibody affinity measurement.Biacore T100 measures people Siglec-8ECD albumen is to mice, the chimeric and combination of humanization anti-Siglec-8 antibody.According to manufacturer on CM5 chip Scheme (Biacore, GE) immobilization capture antibodies (Goat anti human Fc and the goat from Jackson Immunoresearch Anti-mouse Fc).With anti-human antibody's immobilization flow chamber 1,2 and 3, and with anti-mouse antibody immobilization flow chamber 4.In 25 DEG C with 30 The flow velocity of μ l/min is measured method.Measuring buffer is 20mM Tris-HCl pH 8.3,150mM sodium chloride, and 0.05% gathers Pyrusussuriensis ester 20,10% glycerol, 0.1%BSA, prepares in ultra-pure water.Dimer Siglec-8 is diluted (logical in measuring buffer Cross size exclusion chromatography and remove contaminant monomers and oligomer Siglec-8), from 15nM to 1.88pM, 2 times of dilutions.Capture antibodies is extremely The change of about 120RU.Carry out efficiently injection in 6 minutes, be followed by 120 minutes dissociating.By 50mM glycine pH 1.5 recovery stream Dynamic room.Using empty with reference to room with repeatedly mensuration buffer injection is as dual blank, and by 1:1 overall situation fitting parameter analysis result.

Measure Mus 2E2 and the affinity of chimeric 2E2 antibody, be 28pM and 16pM (table 8) respectively.The affinity of humanized antibody Power, HEKA is 17pM and HEKF is 21pM, and assignor source successfully reserves and strengthens combination activity.

Table 8: mice, chimeric and humanized antibody affinity measures

Antibody	ka(1/Ms)	kd(1/s)	KD(pM)
				Mice 2E2	5.56E+05	1.54E-05	28
Chimeric 2E2	8.51E+05	1.32E-05	16
				HEKA	6.38E+05	1.11E-05	17
HEKF	6.78E+05	1.40E-05	21

Affinity of antibody mensuration is carried out also by biosphere interferometric method (ForteBio).At ForteBio Octet Red Mice, the chimeric and humanization anti-Siglec-8 Fab fragments combination to people's Siglec-8 protein is measured on 384.Surveying Determine in buffer the RPM with 1000 and be measured method in 25 DEG C.From stock solution (Biacore BR-10670, ForteBio18- 132) preparation HBS buffer containing 1x ForteBio Kinetics buffer in ultra-pure water.Dilute in measuring buffer Fab fragment (digests antibody with Thermo-Pierce immobilized papain in accordance with the description of manufacturer), from 50nM extremely 1.56nM, 2 times of dilutions.With 100nM Siglec-of immobilization band Fc label on anti-human seizure sensor in measuring buffer The change of 8 protein 3 minutes in terms of nm about 1.2.Carry out 2 minutes combining, be followed by 10 minutes dissociating.With empty with reference to AHC Sensor is as blank, and uses 1:1 overall situation fitting parameter analysis result in ForteBio analyzes software.

Measure Mus 2E2 and the affinity of chimeric 2E2Fab fragment, be 536pM and 585pM (table 9) respectively.Humanized antibody Affinity, HEKA is 464pM and HEKF is 592pM, and assignor source successfully reserves the combination of both humanized antibodies and lives Property.HEKA has the unit price affinity to Siglec-8 higher than mice and chimeric 2E2 in this algoscopy.Humanized antibody Variant, the affinity of HEKAmut and HEKFmut is also in picomolar range, and KD is 902pM and 1160pM respectively.

Table 9: mice, chimeric and humanized antibody affinity measures

Antibody	kon(1/Ms)	kdis(1/s)	KD(pM)
				Mice 2E2	1.14E+06	6.12E-04	536
Chimeric 2E2	9.51E+05	5.56E-04	585
				HEKA	1.04E+06	4.82E-04	464
HEKF	9.20E+05	5.45E-04	592
				HEKAmut	7.26E+05	6.55E-04	902
HEKFmut	4.45E+05	5.16E-04	1160

The dissolubility of humanization candidate antibodies

(Amicon 30K 4mL, 4000g 5min-concentrate for the first time to use centrifugal filter device；Amicon Ultra 0.5mL 3K, 14000g-subsequent concentration) the chimeric and candidate antibodies of sequential concentration purification, and measure concentration in each step. In the case of not precipitation, all samples concentrates the most up to 21-24 times, and is tested by ELISA, shows that none loses knot Close the effect of Siglec-8.The tendency that antibody does not precipitate in the concentration of the most at least 25mg/mL.Specifically, dissolubility, Ch2E2 is at least 18mg/mL, and HEKA is at least 25mg/mL, and HEKF is at least 8mg/mL, and HEKAmut is at least 29mg/mL, and HEKFmut is at least 17mg/mL.

The gathering of humanization candidate antibodies

Filtered sample is to remove any salt or protein precipitation before analysis, and again measures concentration.Then at HPLC They are injected in size-exclusion column with 0.4mL/min by system, and are definitely divided to measure by multi-angle light-scattering analysis Son amount, and detection of aggregation.All variants are displayed without the sign assembled, and average molecular weight range is 134.9-138.2kDa, this It it is IgG monomer desired extent (table 10) in the analysis.

All samples is all monodispersed (Mw/Mn < 1.05).But, distributional analysis figure show glycosylation variants (ch2C4, HEKA and HEKFMut) existence.Distributional analysis figure also shows the existence of about 105-120kDa kind in all samples, and this may It is the antibody or hypo-glycosylated variant decomposed.Mass recovery is 82.9-102.8% (calculating quality to plastic injection quality), this instruction Preferably Protein Recovery, and sample is not the most bonded on post or contains the insoluble aggregates that can be retained by guard column. In a word, any anti-Siglec-8 antibody samples that data instruction is analyzed significantly assembles (table 10).

Table 10: the chimeric and analysis of agglomeration of humanization variant antibodies

The freeze-thaw stability of humanization candidate antibodies

Make the chimeric ch2C4 antibody of purification, humanization HEKA and HEKF antibody, and humanization HEKAmut and HEKFmut Antibody variants stands-20 DEG C and reaches 60 minutes, melts in room temperature, and is used for ELISA algoscopy with the EC80 concentration of every kind of candidate. HEKA shows the highest stability (Fig. 6) in this algoscopy.

The ADCC activity of non-defucosylated antibody

Material

RBC lysis buffer (10X RBC lysis buffer): be diluted to 1X, such as manufacturer (eBioscience, 00- 4300-54) instruct.

PBS: without Ca²⁺/Mg²⁺DPBS (Hyclone, SH30028.02).

The RPMI-1640 (Invitrogen) containing 10%FBS of RPMI: aseptic filtration completely.

96 hole U base plates (Falcon, 353077).

LDH algoscopy: CytoTox 96 non-radioactive cell toxicity assay (Promega, G1780)

FIX buffer: containing the PBS of 1-4% paraformaldehyde.By PBS (Electron Microscopy Diatom, In 50-980-488), dilution is prepared from 16% paraformaldehyde (EM level, without methanol).

Method

In order to test anti-Siglec-8 antibody to eosinophilic ADCC and apoptosis activity, by fresh peripheral blood leukocytes (PBL) incubation together with chimeric and mice 2E2 antibody.Low fucose is fitted together to 2E2IgG1 antibody and shows the strongest thin addicted to eosin Born of the same parents kill and have and are fitted together to the highest effect of 2E2IgG1 than fucosylation, with the ADCC of the antibody of low fucose form Activity higher consistent (Fig. 7).

In order to assess anti-Siglec-8 antibody activity in total peripheral blood leucocyte, by standard method from results less than 24 The donor's blood hour collected obtains PBL, and resuspension in complete RPMI culture medium.To cell counting, at complete RPMI Culture medium adjusts to 10x 10⁶/ mL, and distribute in aseptic 96 hole U base plates with 100 μ L/ holes.With 0.0001ng/mL to 10 μ The concentration of g/mL adds anti-Siglec-8 antibody.Plate is centrifuged 1 minute with 200g, and at humidification 37 DEG C, 5%CO₂The middle temperature of incubator Educate ＞ 4 hours.Cut down with assessment eosinocyte and basophil by flow cytometry assessment cell mass, such as, use table Reagent shown in 11, and assessed by flow cytometry.CCR3 can be used positive, CD16 negative granules cell (high sidescattering) Removal detect eosinophilic abatement.Such as can measure basophilia by the positive low sidescattering cell of analysis CCR3 Cell counting.

Table 11: reagent

The ability apoptosis-induced in the eosinocyte of purification in order to assess anti-Siglec-8 antibody, uses from results Play the fresh buffy coat collected less than 24 hours autoblood samples or equivalent blood products.Follow the description of manufacturer (Miltenyi eosinocyte separating kit, 130-092-010) carries out eosinophilic purification.Cultivate at complete RPMI With 1x 10 in base⁶The eosinocyte of/mL resuspension purification, and in IL-5 (with the concentration of about 1ng/mL to about 50ng/mL) Exist or the lower overnight incubation of disappearance.Next day, gathered in the crops the eosinocyte of cultivation by repeated washing plate or flask.By cell with 200-400g is centrifugal less than 10 minutes, and with 1x 10 in complete RPMI culture medium⁶/ mL resuspension.By eosinocyte with 100 μ L/ holes are distributed in aseptic 96 hole U base plates.The 2X reagent prepared in complete RPMI culture medium by 100uL adds to each Hole, and prepare diluent as described above.Plate is centrifuged 1 minute with 200g, and at humidification 37 DEG C, 5%CO₂Incubation in incubator >= 4 hours.Annexin V dyeing is implemented according to the description of manufacturer, and by flow cytometry apoptosis and non-viable non-apoptotic cell.

In order to assess anti-Siglec-8 antibody ADCC on the mastocyte separated and apoptosis activity, according to delivered Scheme (Guhl et al., Biosci.Biotechnol.Biochem., 2011,75:382-384；Kulka et al.,In Current Protocols in Immunology, 2001, (John Wiley&Sons, Inc.)) fertile from people separate tissue people Maxicell, or from human hematopoietic stem cell differentiation of human mastocyte, such as such as Yokoi et al., J Allergy Clin Immunol., described in 2008,121:499-505.With 1x 10⁶/ mL in complete RPMI culture medium in aseptic 96 hole U base plates The mastocyte of resuspension purification, and at anti-Siglec-8 antibody (with scope between 0.0001ng/ml and 10 μ g/ml Concentration) exist or lack lower incubation 30 minutes.With or without NK cell (NK) cell of purification or the situation of fresh PBL Lower by sample incubation 4 to 16 hours again to induce ADCC.Use the detection mastocyte (CD117 and Fc ε R1) puted together of fluorescence and The antibody of annexin V and 7AAD (distinguishing live and cell that is dead or that dying) passes through to wither by flow cytometry Die or the cell killing of ADCC.Annexin V and 7AAD dyeing is implemented according to the description of manufacturer.

The assessment of the eosinocyte abatement activity of humanized antibody in vitro

To humanized antibody assessment, they induce what Siglec-8 mediate to cut down thin addicted to eosin from normal human blood in vitro The ability of born of the same parents, compares with Mus 2E2 antibody.

[(it is supplemented with RPMI-1640 culture medium (Invitrogen, the product of 10% hyclone in complete RPMI culture medium Catalog number (Cat.No.) A10491-01))] in resuspension after harvesting less than 24 hours collect the peripheral blood from people's donor's blood white Cell (PBL).It is being supplemented with the complete of 50ng/ml recombined human IL-5 (R&D Systems, catalog number 205-IL-025) Cell is adjusted to 10 by RPMI culture medium⁷Every mL, and distribute in aseptic 96 hole U base plates with 100 μ L/ holes.With scope it is 0.1pg/mL to 10 μ g/mL (i.e. 1pg/mL, 10pg/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 0.1 μ g/mL, 1 μ g/mL, and 10 μ g/mL) concentration add anti-Siglec-8 antibody to measure dose response and to provide maximum eosinocyte abatement 50% Concentration (EC₅₀).Plate is centrifuged 1 minute with 200g, and at humidification 37 DEG C, 5%CO₂Incubation 16 hours in incubator.PBL is with anti- Siglec-8 antibody together in the presence of IL-5 incubation within 16 hours, make the apoptosis sensitization that Siglec-8 is mediated by eosinocyte.Logical Overflow-type cell art assessment cell mass is with assessment eosinocyte abatement.By going of CCR3 positive particle (high sidescattering) cell Remove and detect eosinocyte abatement.

In addition to HEKAmut IgG1 antibody, the humanized antibody of each test is for the abatement display of people's eosinocyte Equivalent or effect (the lowest EC of rising compared with mice 2E2 antibody₅₀) (table 12).Antibody is for abatement people's eosinocyte Effect do not rely on isotype, because HEKA IgG1 antibody shows similar effect with HEKA IgG4 antibody.

Table 12: humanization anti-Siglec-8 antibody cuts down eosinophilic effect in vitro

Antibody	Isotype	Average EC of eosinocyte abatement50(ng/ml)
			2E2	IgG1	6.8
HEKA	IgG1	4.3
			HEKA	IgG4	3.9
HEKAmut	IgG1	43.3
			HEKF	IgG1	6.7
HEKFmut	IgG1	6.9

Average EC₅₀The eosinocyte from 2 independent algoscopys is indicated to cut down the maximum antibody concentration of average half needed.

People's mastocyte that the anti-Siglec-8 antibody of active isotype can induce ADCC to mediate in vitro and in vivo Kill.

In order to generate the humanization anti-Siglec-8 antibody with the receptor-mediated ADCC activity of strong Fc, from fucose HEKA IgG1 antibody is expressed with life by the Chinese hamster ovary celI system (Lonza, Potelligent CHOK1SV cell) of based transferase-8 defect Become the antibody (antibody of the most non-fucosylation) with the carbohydrate lacking α 1,6-fucose.Such as institute in embodiment 3 State, generate the Mus monoclonal anti from the Mus IgG2a isotype of HEKA IgG1 antibody recognition different Siglec-8 extracellular region Siglec-8 antibody (i.e. 1C3 antibody).Chimeric 1H10 antibody contains V district and human IgG1's Kappa perseverance of mouse monoclonal antibody 1H10 Determine district.The people's 293TS expression of cell lines cultivated in the presence of comfortable 10 μMs of several husband's alkali (Kifunensine) is fitted together to 1H10 antibody with life Become low fucose antibody, and carry out purification by protein A affinity chromatography.

HEKA IgG1 and the chimeric 1H10 low fucose antibody induction NK assessing non-fucosylation in vitro is cell-mediated The ability of the ADCC activity for people's mastocyte.Being transplanted by lavation has the immunodefiiciency NSGS of human hematopoietic stem cell little The abdominal cavity of Mus separates primary people's mastocyte.By mastocyte and the HEKA IgG1 antibody of the 10 non-fucosylations of μ g/ml, Chimeric 1H10 low fucose antibody, HEKA IgG4 antibody or isotype controls human IgG1's antibody, and with the people CD56 of purification⁺ CD16⁺NK effector lymphocyte incubation 48 hours together, effector lymphocyte is 10.75:1 to target cell (E:T) ratio.Use CytoTox 96 Cytotoxicity assay test kit (Promega, catalog number G1780) measures ADCC activity by LDH release.Non-fucose The people that the HEKA IgG1 antibody of base and chimeric 1H10 low fucose antibody induce significant ADCC to mediate after 48 hrs is loose Cell killing (Fig. 8 A).Being discharged by the LDH of the anti-Siglec-8 antibody induction of non-fucosylation or low fucose is to use The 38-53% of the maximum LDH release that cracked solution (Promega, catalog number G1821) is induced.

People Siglec-8 on the surface of mastocyte, eosinocyte and basophil selective expression turn base The ability of Siglec-8 Positive Mast Cells is cut down in vivo because mouse model is assessed anti-Siglec-8 antibody.Pass through intraperitoneal Injecting 100 μ g HEKA IgG4 antibody, the humanized antibody of the non-fucosylation of HEKA IgG1, (Mus IgG2a is same for Mus 1C3 antibody Kind of type) or human IgG1's Isotype control antibodies process mice twice.It is separated by 48 hours and uses twice peritoneal injection, and second Secondary injection separates peritoneal mast cells by lavation peritoneum in latter 48 hours.The HEKA IgG1 antibody of non-fucosylation and Mus 1C3 antibody is used and is caused significant peritoneal mast cells to be cut down.In contrast, HEKA IgG4 antibody does not show significant hypertrophy Cell depletion, in indication body, mastocyte abatement requires active isotype (Fig. 8 B).These results prove active isotype (Mus IgG2a isotype or the isotype of the non-fucosylation of human IgG1) two kinds of the zones of different for Siglec-8 extracellular domain Different anti-Siglec-8 antibody can cut down Siglec-8 Positive Mast Cells in vivo.

These results are unexpected, because Siglec-8 has been described quick internalization, are therefore not suitable for induction ADCC activity.See O ' Reilly et al., Trends Pharmacol Sci., 2009,30 (5): 240-248.

The anti-Siglec-8 of humanization suppress that IgE induces in vivo by the passive cutaneous anaphylaxis, PCA of people's mast cell mediated

The immune deficient mice that can generate abundant people's mastocyte after Transplanted Human hematopoietic stem cell (HSC) has been remembered Carry (Tanaka et al., J Immunol., 2012,188 (12): 6145-55).It is referred to as NSGS (The Jackson Laboratory) mouse species is that non-obese diabetes/severe combined immunodeficient (NOD SCID) mice has IL-2 and is subject to Body gamma chain gene deletes the derivant of (NSG mice).Additionally NSGS mice is carried out 3 kinds of human cell factor (stem cell factors [SCF], IL-3, and GM-CSF) transgenic with promote human hematopoietic stem cell transplant.After NSGS mice is transplanted, people CD34⁺Carefully Born of the same parents generate people's mastocyte of people's eosinocyte and increased number.Transplant the both cell types in NSGS mice all with The horizontal expression Siglec-8 being on close level on the corresponding cell type of human peripheral and separate tissue.So, these are little Mus provides a kind of tempting model for the activity assessing the most anti-Siglec-8 antibody on human cell.

In order to assess the impact in vivo on mast cell activity of the anti-Siglec-8 antibody, build in humanization NSGS mice The ear swelling model of vertical a kind of IgE mediation.In this model, induce models of passive skin irritability (PCA) (a kind of I type hypersensitization as follows Property reaction), will be injected in an ear by specific monoclonal anti hapten IgE (anti-NP IgE), after 24 hours, systemic injection is sewed Conjunction has haptenic bovine serum albumin (NP-BSA).The inosculating antibody NP IgE with people ε constant region is used to guarantee to generate pin To haptenic people's mastocyte specificly-response, and by the change of ear thickness measure immediately with edema response in late period.

Before algoscopy, 8-12 week is to NSGS mice Transplanted Human CD34⁺HSC.With the dosage Intradermal of 100ng in mice Injection has the chimeric monoclonal anti-NP IgE of human constant region and enters with sensitization people but non-mouse skin mastocyte in auris dextra, and skin Interior injection PBS enters in left ear.After 24 hours, induce PCA by intravenous injection 0.5mg NP-BSA.Quick with anti-NP IgE Change first 24 hours or by intravenous injection, mice be administered 0.1mg anti-Siglec-8 antibody (i.e. HEKA in after sensitization 2 hours IgG4 antibody) or human IgG 4 Isotype control antibodies.In evening to inducing latter 4 hours and the induction point in time measurement ear of latter 24 hours Thickness is to measure the response of early and late ear swelling respectively.

HEKA IgG4 antibody prevents in this PCA In vivo model or suppresses both early and late skin allergy reactions (Fig. 9).In this model, early reaction depends on mastocyte threshing and histamine release, and late phase response depends on loose thin The medium of intracrine de novo synthesis, including cytokine, and eosinocyte and basophil infiltrate.HEKA IgG4 resists Body also prevention or suppression when being administered with after first 24 hours of anti-NP IgE sensitization or sensitization 2 hours in humanization NSGS mice PCA response (Fig. 9).The adverse effect of antibody treatment is not observed during these experimentations.

Embodiment 3: the generation of mouse-anti Siglec-8 antibody and sign.

The extracellular region of Siglec-8 is made up of three immunoglobulin-like territories: the N end V-set of a unique binding partner Domain (domain 1), is followed by two C-set domains (domain 2 and 3).Antibody 1C3 is for recombined human Siglec-8 What (SEQ ID NO:74) extracellular domain generated has IgG2a heavy chain and the mouse monoclonal antibody of Kappa light chain.Monoclonal antibody 1H10 and 4F11 is the Mus IgG1 heavy chain and Kappa light chain generated for recombined human Siglec-8 (SEQ ID NO:74) extracellular domain Antibody.It is shown in Table 13.These antibody be from for combine from people (SEQ ID NO:74) and non-human primates (SEQ ID NO: 118) the hybridoma Screening and Identification of the antibody of restructuring Siglec-8 sequence.

Table 13: from the aminoacid sequence of the HVR of Mus 1C3,1H10, and 4F11 antibody

In order to identify the region of the epi-position comprising anti-Siglec-8 antibody, contain from expressing cho cell purification and be fused to The fusion protein of each Siglec-8 extracellular domain of people Ig-Fc.ELISA algoscopy for measuring antibodies uses Containing people's domain 1 (SEQ ID NO:115), domain 1 and 2 (SEQ ID NO:116), or domain 1,2, and 3 (SEQ ID NO:117) fusion protein.In some are tested, the specificity of assessment antibody on human Siglec-8, and containing from green baboon The Siglec-8 extracellular domain 1,2 of (Papio anubis), and 3 (SEQ ID NO:118) (National Center for Biotechnology Information canonical sequence XP_009193370.1) fusion protein compare.

For antibodies algoscopy, by elisa plate (MaxiSorp；Nunc) with 0.2 μ g/ml fusion protein in 4 DEG C of bags By overnight, and close 1 hour in room temperature with the PBS containing 2%BSA.Add 1 μ g/ml antibody, and by plate in incubation at room temperature 2 hours. After clean plate, add that horseradish peroxidase puts together two resist, and by plate incubation 1 hour.The two of humanized antibody is resisted and is Anti-human H+L HRP (Jackson ImmunoResearch, catalog number 709-035-149), or for mouse antibodies Two anti-be anti-mouse H+L HRP (Jackson ImmunoResearch, catalog number 715-035-151).Plate is used at the bottom of TMB Thing (Sigma, catalog number T0440-1L) develops the color.

Mus 2E2 antibody and HEKA IgG1 antibody binding domain 1 fusion protein, the epi-position of instruction both antibody is positioned at N In end ligand binding domains (table 14).On the contrary, Mus 1C3 antibody binding domain 1 and domain 2 fusion protein but do not represent Detected combination to domain 1 fusion protein, indicates the epi-position (table 14) in domain 2 of this antibody.

Table 14: the combination of the epi-position in anti-Siglec-8 antibody on human Siglec-8

Mus 4F11 and 1H10 antibodies people Siglec-8 and the prediction Siglec-from green baboon (Papio anubis) 8 protein sequences (National Center for Biotechnology Information canonical sequence XP_ 009193370.1).In the ELISA of domain fusion protein screens and in the western blot of reproducibility PAGE gel On, 4F11 identifies the linear epitope in people's Siglec-8 domain 1, and 1H10 identifies and includes in people's Siglec-8 domain 3 The linear epitope of sequence.1C3 fails to see the degeneration sequence in others' Siglec-8 domain 2, indicates it to identify comformational epitope.? In the presence of 50ng/ml IL-5, antibody 4F11 and 1H10 shows the strong abatement eosinocyte from human peripheral leucocytes, EC₅₀It is 5.9 and 41ng/ml (tables 15) respectively.Mus 1C3 antibody specific to people Siglec-8 and Mus 2E2 antibody not with baboon Siglec-8 cross reaction.

Table 15: combination and the antibody on human of the epi-position in anti-Siglec-8 antibody on human or baboon Siglec-8 are thin addicted to eosin The abatement activity of born of the same parents

Average EC₅₀The eosinocyte from 2 independent algoscopys is indicated to cut down the maximum antibody concentration of average half needed.

By the eosinophilic combination of Flow Cytometry Assay antibody on human and baboon.With the anti-Siglec-8 of saturation capacity Prepared by monoclonal antibody 2E2,1C3, and 1H10 or mouse IgG 1 Isotype control antibodies labelling people or baboon peripheral blood leucocyte Thing.Anti-Siglec-8 antibody is manifested by anti-mouse IgG H+L AlexaFluor 647 2 is anti-.Eosinocyte is to use pin The primates cross reactivity monoclonal antibody of CD49d and CD16 is identified together with the scattering of high granule.Mus 1H10 antibody In conjunction with baboon and people's eosinocyte, and mice 2E2 and 1C3 antibodies people's eosinocyte but not with baboon eosinocyte Cross reaction (Figure 10).These results are unexpected, because other Monoclonal mouse combining people Siglec-8 resists Siglec-8 antibody demonstrates nonrecognition non-human primates Siglec-8.See Hudson et al., J.Clin.Immunol., 2011,31(6):1045-53。

Sequence

The aminoacid sequence of mice 2E2 heavy chain variable domain

QVQLKESGPGLVAPSQSLSITCTVSGFSLTIYGAHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQ VFLKINSLQTDDTALYYCARDGSSPYYYSMEYWGQGTSVTVSS(SEQ ID NO:1)

The aminoacid sequence of 2E2 RHA heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVSVIWAGGSTNYNSALMSRFTISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSS (SEQ ID NO:2)

The aminoacid sequence of 2E2 RHB heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAVSGFSLTIYGAHWVRQAPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSS (SEQ ID NO:3)

The aminoacid sequence of 2E2 RHC heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAVSGFSLTIYGAHWVRQAPGKGLEWVSVIWAGGSTNYNSALMSRFTISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSS (SEQ ID NO:4)

The aminoacid sequence of 2E2 RHD heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWLSVIWAGGSTNYNSALMSRFTISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSS (SEQ ID NO:5)

The aminoacid sequence of 2E2 RHE heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSS (SEQ ID NO:6)

The aminoacid sequence of 2E2 RHF heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVSVIWAGGSTNYNSALMSRLTISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSS (SEQ ID NO:7)

The aminoacid sequence of 2E2 RHG heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVSVIWAGGSTNYNSALMSRFSISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSS (SEQ ID NO:8)

The aminoacid sequence of 2E2 RHA2 heavy chain variable domain

QVQLQESGPGLVKPSETLSLTCTVSGGSISIYGAHWIRQPPGKGLEWIGVIWAGGSTNYNSALMSRVTISVDTSKNQ FSLKLSSVTAADTAVYYCARDGSSPYYYSMEYWGQGTLVTVSS (SEQ ID NO:9)

The aminoacid sequence of 2E2 RHB2 heavy chain variable domain

QVQLQESGPGLVKPSETLSLTCTVSGFSLTIYGAHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKNQ VSLKLSSVTAADTAVYYCARDGSSPYYYSMEYWGQGTLVTVSS(SEQ ID NO:10)

The aminoacid sequence of 2E2 RHE S-G mutant heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYGMEYWGQGTTVTVSS(SEQ ID NO:11)

The aminoacid sequence of 2E2 RHE E-D heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYSMDYWGQGTTVTVSS(SEQ ID NO:12)

The aminoacid sequence of 2E2 RHE Y-V heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYSMEVWGQGTTVTVSS(SEQ ID NO:13)

The aminoacid sequence of 2E2 RHE triple mutants heavy chain variable domain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYGMDVWGQGTTVTVSS(SEQ ID NO:14)

The aminoacid sequence of mice 2E2 light-chain variable domain

QIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRM EAEDAATYYCQQRSSYPFTFGSGTKLEIK(SEQ ID NO:15)

The aminoacid sequence of 2E2 RKA light-chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSSYPFTFGPGTKLDIK(SEQ ID NO:16)

The aminoacid sequence of 2E2 RKB light-chain variable domain

EIILTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLWIYSTSNLASGVPARFSGSGSGTDYTLTISSL EPEDFAVYYCQQRSSYPFTFGPGTKLDIK(SEQ ID NO:17)

The aminoacid sequence of 2E2 RKC light-chain variable domain

EIILTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSSYPFTFGPGTKLDIK(SEQ ID NO:18)

The aminoacid sequence of 2E2 RKD light-chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLWIYSTSNLASGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSSYPFTFGPGTKLDIK(SEQ ID NO:19)

The aminoacid sequence of 2E2 RKE light-chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGVPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSSYPFTFGPGTKLDIK(SEQ ID NO:20)

The aminoacid sequence of 2E2 RKF light-chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDYTLTISSL EPEDFAVYYCQQRSSYPFTFGPGTKLDIK(SEQ ID NO:21)

The aminoacid sequence of 2E2 RKG light-chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWYQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSSYPFTFGPGTKLDIK(SEQ ID NO:22)

The aminoacid sequence of 2E2 RKA F-Y mutant light-chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSSYPYTFGPGTKLDIK(SEQ ID NO:23)

The aminoacid sequence of 2E2 RKF F-Y mutant light-chain variable domain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDYTLTISSL EPEDFAVYYCQQRSSYPYTFGPGTKLDIK(SEQ ID NO:24)

HEKA IgG1 heavy chain and the aminoacid sequence of HEKF IgG1 heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 75)

The aminoacid sequence of HEKA Kappa light chain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSSYPFTFGPGTKLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:76)

The aminoacid sequence of HEKF Kappa light chain

EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDYTLTISSL EPEDFAVYYCQQRSSYPFTFGPGTKLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:77)

The aminoacid sequence of IgG1 CH

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG(SEQ ID NO:78)

The aminoacid sequence (IgG4 contains S228P sudden change) of IgG4 CH

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLG(SEQ ID NO:79)

The aminoacid sequence of Ig Kappa constant region of light chain

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:80)

Mus 2C4 and the aminoacid sequence of 2E2 IgG1 heavy chain

QVQLKRASGPGLVAPSQSLSITCTVSGFSLTIYGAHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKS QVFLKINSLQTDDTALYYCARDGSSPYYYSMEYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF PEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICT VPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMH QDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQP AENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG(SEQ ID NO:81)

The aminoacid sequence of Mus 2C4 Kappa light chain

EIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRM EAEDAATYYCQQRSSYPFTFGSGTKLEIKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:82)

The aminoacid sequence of Mus 2E2 Kappa light chain

QIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRM EAEDAATYYCQQRSSYPFTFGSGTKLEIKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:83)

The aminoacid sequence of chimeric 2C4 and 2E2 IgG1 heavy chain

QVQLKRASGPGLVAPSQSLSITCTVSGFSLTIYGAHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKS QVFLKINSLQTDDTALYYCARDGSSPYYYSMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 84)

The aminoacid sequence of chimeric 2C4 Kappa light chain

EIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRM EAEDAATYYCQQRSSYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:85)

The aminoacid sequence of chimeric 2E2 Kappa light chain

QIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRM EAEDAATYYCQQRSSYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:86)

The aminoacid sequence (IgG4 contains S228P sudden change) of HEKA IgG4 heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNT VYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:87)

(residue being underlined comprises according to Chothia numbering the aminoacid sequence of mice 1C3 heavy-chain variable domains CDR H1 and H2 of mode)

EVQVVESGGDLVKSGGSLKLSCAASGFPFSSYAMSWVRQTPDKRLEWVAIISSGGSYTYYSDSVKGRFTISRDNAKN TLYLQMSSLKSEDTAMYYCARHETAQAAWFAYWGQGTLVTVSA(SEQ ID NO:106)

(residue being underlined comprises according to Chothia numbering side the aminoacid sequence of mice 1H10 heavy chain variable domain CDRH1 and H2 of formula)

EVQLQQSGAELVRPGASVKLSCTASGFNIKDYYMYWVKQRPEQGLEWIGRIAPEDGDTEYAPKFQGKATVTADTSSN TAYLHLSSLTSEDTAVYYCTTEGNYYGSSILDYWGQGTTLTVSS(SEQ ID NO:107)

(residue being underlined comprises according to Chothia volume the aminoacid sequence of mice 4F11 heavy-chain variable domains CDR H1 and H2 of number mode)

QVQLQQSGAELVKPGASVKISCKASGYAFRSSWMNWVKQRPGKGLEWIGQIYPGDDYTNYNGKFKGKVTLTADRSSS TAYMQLSSLTSEDSAVYFCARLGPYGPFADWGQGTLVTVSA(SEQ ID NO:108)

The aminoacid sequence of mice 1C3 light-chain variable domain

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLAYGVPARFSGSGSGTSYSLTISSM EAEDAATYYCQQWSSNPPTFGGGTKLEIK(SEQ ID NO:109)

The aminoacid sequence of mice 1H10 light variable domains

DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQQKPDGTVKLLIYFTSRLHSGVPSRFSGSGSGTDYSLTISN LEQEDIATYFCQQGNTLPWTFGGGTKLEIK(SEQ ID NO:110)

The aminoacid sequence of mice 4F11 light-chain variable domain

QIVLTQSPAIVSASPGEKVTMTCSASSSVSYMYWYQQRPGSSPRLLIYDTSSLASGVPVRFSGSGSGTSYSLTISRI ESEDAANYYCQQWNSDPYTFGGGTKLEIK(SEQ ID NO:111)

The aminoacid sequence of people's Siglec-8 domain 1

MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQ GRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRP(SEQ ID NO: 112)

The aminoacid sequence of people's Siglec-8 domain 2

DILILGTLESGHSRNLTCSVPWACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVT TTSTVRLDVS(SEQ ID NO:113)

The aminoacid sequence of people's Siglec-8 domain 3

YPPWNLTMTVFQGDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVHVR DEGEFTCRAQNAQGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGG(SEQ ID NO:114)

The aminoacid sequence of people's Siglec-8 domain 1 fusion protein

MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQ GRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPIEGRSDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 115)

The aminoacid sequence of people's Siglec-8 domain 1 and 2 fusion protein

MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQ GRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHS RNLTCSVPWACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSIEG RSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:116)

People's Siglec-8 domain 1,2, and the aminoacid sequence of 3 fusion protein

MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQ GRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHS RNLTCSVPWACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPP WNLTMTVFQGDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVHVRDEG EFTCRAQNAQGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGIEGRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:117)

Baboon Siglec-8 domain 1,2, and the aminoacid sequence of 3 fusion protein

MEGDRKYGDGYLLQVQELVTVQEGLCVHVPCSFSYPKDDWTYSDPVHGYWFRAGDRPYQEAPVATNNPDTEVQAETQ GRFQLLGDRWSNDCSLSINDARKGDEGSYFFRLERGRMKWSYKSQLNYKAKQLSVFVTALTQRPDILIQGTLESGHP RNLTCSVPWACEQRMPPMISWIGTSVSSLGPITARFSVLTLIPKPQDHGTSLTCQVTLPGTGVTTTRTVQLDVSYPP WNLTVTVFQGDDTASTALGNGSSLSVLEGQSLRLVCAVDSNPPARLSWTRGSLTLCPSQPWNPGLLELLRVHVKDEG EFTCQAENPRGSQHISLSLSLQNEGTGTARPVSEVTLAAVGGIEGRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:118)

Baboon Siglec-8 domain 1,2, and the aminoacid sequence of 3

MEGDRKYGDGYLLQVQELVTVQEGLCVHVPCSFSYPKDDWTYSDPVHGYWFRAGDRPYQEAPVATNNPDTEVQAETQ GRFQLLGDRWSNDCSLSINDARKGDEGSYFFRLERGRMKWSYKSQLNYKAKQLSVFVTALTQRPDILIQGTLESGHP RNLTCSVPWACEQRMPPMISWIGTSVSSLGPITARFSVLTLIPKPQDHGTSLTCQVTLPGTGVTTTRTVQLDVSYPP WNLTVTVFQGDDTASTALGNGSSLSVLEGQSLRLVCAVDSNPPARLSWTRGSLTLCPSQPWNPGLLELLRVHVKDEG EFTCQAENPRGSQHISLSLSLQNEGTGTARPVSEVTLAAVGG(SEQ ID NO:119)

Claims (109)

1. combine a humanized antibody of people Siglec-8, wherein this humanized antibody binding affinity to people Siglec-8 Or affinity is higher to binding affinity or the affinity of people Siglec-8 than antibody 2E2 or 2C4.

2. combining a humanized antibody of people Siglec-8, wherein this antibody is at hot transfer assay method (thermal shift Assay) there is in the Tm of at least about 70 DEG C.

3. the antibody of claim 2, wherein this antibody has at least about 70 DEG C at least about 72 DEG C in hot transfer assay method Tm。

4. the antibody of any one of claim 1-3, wherein this antibody comprises variable region of heavy chain and variable region of light chain, wherein this heavy chain Variable region comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises the amino of SEQ ID NO:62 The HVR-H2 of acid sequence, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:63；And/or wherein this light chain can Becoming district and comprise the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, (ii) comprises the aminoacid of SEQ ID NO:65 The HVR-L2 of sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:66.

5. the antibody of any one of claim 1-4, wherein this antibody comprises the heavy chain of aminoacid sequence of SEQ ID NO:6 Variable region；And/or comprise the variable region of light chain of aminoacid sequence selected from SEQ ID NO:16 or 21.

6. the antibody of any one of claim 1-5, wherein this antibody comprises the heavy chain Fc district in human IgG Fc district.

7. the antibody of claim 6, wherein this human IgG Fc district comprises human IgG1 or human IgG 4.

8. the antibody of claim 7, wherein this human IgG 4 comprises amino acid replacement S228P, and wherein amino acid residue be according to EU index number in Kabat.

9. the antibody of any one of claim 1-7, wherein this antibody comprises the weight of aminoacid sequence of SEQ ID NO:75 Chain；And/or comprise the light chain of aminoacid sequence selected from SEQ ID NO:76 or 77.

10. the antibody of any one of claim 1-7, wherein this antibody comprises the weight of aminoacid sequence of SEQ ID NO:87 Chain；And/or comprise the light chain of the aminoacid sequence of SEQ ID NO:76.

The antibody of 11. any one of claim 1-9, this antibody the most engineered is situated between to improve antibody dependent cellular Cytotoxicity (ADCC) activity led.

The antibody of 12. any one of claim 1-11, wherein the heavy chain at least one of this antibody or two are non-fucosylations 's.

13. 1 kinds of humanized antibodies combining people Siglec-8, wherein this antibody comprises:

A () variable region of heavy chain and variable region of light chain, wherein this variable region of heavy chain comprises the aminoacid that (i) comprises SEQ ID NO:61 The HVR-H1 of sequence, (ii) comprises the HVR-H2 of the aminoacid sequence of SEQ ID NO:62, and (iii) comprises selected from SEQ ID The HVR-H3 of the aminoacid sequence of NO:67-70；And/or wherein this variable region of light chain comprises the ammonia that (i) comprises SEQ ID NO:64 The HVR-L1 of base acid sequence, (ii) comprises the HVR-L2 of the aminoacid sequence of SEQ ID NO:65, and (iii) comprises SEQ ID The HVR-L3 of the aminoacid sequence of NO:71；Or

B () variable region of heavy chain and variable region of light chain, wherein this variable region of heavy chain comprises the aminoacid that (i) comprises SEQ ID NO:61 The HVR-H1 of sequence, (ii) comprises the HVR-H2 of the aminoacid sequence of SEQ ID NO:62, and (iii) comprises SEQ ID NO:63 The HVR-H3 of aminoacid sequence；And/or wherein this variable region of light chain comprises the aminoacid sequence that (i) comprises SEQ ID NO:64 HVR-L1, (ii) comprises the HVR-L2 of the aminoacid sequence of SEQ ID NO:65, and (iii) comprises the ammonia of SEQ ID NO:66 The HVR-L3 of base acid sequence.

The antibody of 14. claim 13, wherein this antibody comprises the weight of the aminoacid sequence selected from SEQ ID NO:11-14 Chain variable region；And/or comprise the variable region of light chain of aminoacid sequence selected from SEQ ID NO:23-24.

15. 1 kinds of humanized antibodies combining people Siglec-8, wherein this antibody comprises selected from SEQ ID NO:2-14's The variable region of heavy chain of aminoacid sequence；And/or comprise the variable region of light chain of aminoacid sequence selected from SEQ ID NO:16-24.

16. 1 kinds of humanized antibodies combining people Siglec-8, wherein this antibody comprises selected from SEQ ID NO:2-10's The variable region of heavy chain of aminoacid sequence；And/or comprise the variable region of light chain of aminoacid sequence selected from SEQ ID NO:16-22.

17. 1 kinds of humanized antibodies combining people Siglec-8, wherein this antibody comprises:

(a) variable region of heavy chain, it comprises:

(1) HC-FR1 of aminoacid sequence selected from SEQ ID NO:26-29 is comprised；

(2) HVR-H1 of the aminoacid sequence of SEQ ID NO:61 is comprised；

(3) HC-FR2 of aminoacid sequence selected from SEQ ID NO:31-36 is comprised；

(4) HVR-H2 of the aminoacid sequence of SEQ ID NO:62 is comprised；

(5) HC-FR3 of aminoacid sequence selected from SEQ ID NO:38-43 is comprised；

(6) HVR-H3 of the aminoacid sequence of SEQ ID NO:63 is comprised；With

(7) HC-FR4 of aminoacid sequence selected from SEQ ID NO:45-46 is comprised,

And/or

(b) variable region of light chain, it comprises:

(1) LC-FR1 of aminoacid sequence selected from SEQ ID NO:48-49 is comprised；

(2) HVR-L1 of the aminoacid sequence of SEQ ID NO:64 is comprised；

(3) LC-FR2 of aminoacid sequence selected from SEQ ID NO:51-53 is comprised；

(4) HVR-L2 of the aminoacid sequence of SEQ ID NO:65 is comprised；

(5) LC-FR3 of aminoacid sequence selected from SEQ ID NO:55-58 is comprised；

(6) HVR-L3 of the aminoacid sequence of SEQ ID NO:66 is comprised；With

(7) LC-FR4 of the aminoacid sequence of SEQ ID NO:60 is comprised.

The humanized antibody of 18. claim 17, wherein this antibody comprises:

(a) variable region of heavy chain, it comprises:

(1) HC-FR1 of the aminoacid sequence of SEQ ID NO:26 is comprised；

(2) HVR-H1 of the aminoacid sequence of SEQ ID NO:61 is comprised；

(3) HC-FR2 of the aminoacid sequence of SEQ ID NO:34 is comprised；

(4) HVR-H2 of the aminoacid sequence of SEQ ID NO:62 is comprised；

(5) HC-FR3 of the aminoacid sequence of SEQ ID NO:38 is comprised；

(6) HVR-H3 of the aminoacid sequence of SEQ ID NO:63 is comprised；With

(7) HC-FR4 of the aminoacid sequence of SEQ ID NO:45 is comprised；

And/or

(b) variable region of light chain, it comprises:

(1) LC-FR1 of the aminoacid sequence of SEQ ID NO:48 is comprised；

(2) HVR-L1 of the aminoacid sequence of SEQ ID NO:64 is comprised；

(3) LC-FR2 of the aminoacid sequence of SEQ ID NO:51 is comprised；

(4) HVR-L2 of the aminoacid sequence of SEQ ID NO:65 is comprised；

(5) LC-FR3 of the aminoacid sequence of SEQ ID NO:55 is comprised；

(6) HVR-L3 of the aminoacid sequence of SEQ ID NO:66 is comprised；With

(7) LC-FR4 of the aminoacid sequence of SEQ ID NO:60 is comprised.

The humanized antibody of 19. claim 17, wherein this antibody comprises:

(a) variable region of heavy chain, it comprises:

(1) HC-FR1 of SEQ ID NO:26 aminoacid sequence is comprised；

(2) HVR-H1 of the aminoacid sequence of SEQ ID NO:61 is comprised；

(3) HC-FR2 of the aminoacid sequence of SEQ ID NO:34 is comprised；

(4) HVR-H2 of the aminoacid sequence of SEQ ID NO:62 is comprised；

(5) HC-FR3 of the aminoacid sequence of SEQ ID NO:38 is comprised；

(6) HVR-H3 of the aminoacid sequence of SEQ ID NO:63 is comprised；With

(7) HC-FR4 of the aminoacid sequence of SEQ ID NO:45 is comprised；

And/or

(b) variable region of light chain, it comprises:

(1) LC-FR1 of the aminoacid sequence of SEQ ID NO:48 is comprised；

(2) HVR-L1 of the aminoacid sequence of SEQ ID NO:64 is comprised；

(3) LC-FR2 of the aminoacid sequence of SEQ ID NO:51 is comprised；

(4) HVR-L2 of the aminoacid sequence of SEQ ID NO:65 is comprised；

(5) LC-FR3 of the aminoacid sequence of SEQ ID NO:58 is comprised；

(6) HVR-L3 of the aminoacid sequence of SEQ ID NO:66 is comprised；With

(7) LC-FR4 of the aminoacid sequence of SEQ ID NO:60 is comprised.

The antibody of 20. any one of claim 13-19, this antibody the most engineered is to improve antibody dependent cellular Cytotoxicity (ADCC) activity of mediation.

The antibody of 21. any one of claim 13-20, wherein the heavy chain at least one of this antibody or two are non-fucosylations 's.

22. 1 kinds of nucleic acid, the antibody of its coding any one of claim 1-21.

23. 1 kinds of carriers, it comprises the nucleic acid of claim 22.

The carrier of 24. claim 23, it is expression vector.

25. 1 kinds of host cells, it comprises the nucleic acid of claim 22.

26. 1 kinds of methods generating antibody, it is included in the host cell cultivating claim 25 under conditions of generating this antibody.

The method of 27. claim 26, it farther includes to reclaim the antibody generated by this host cell.

The 28. anti-Siglec-8 antibody generated by the method for claim 26 or 27.

29. 1 kinds of pharmaceutical compositions, it comprises claim 1-21 and the antibody of 28 any one and pharmaceutically acceptable supporting agent.

The compositions of 30. 1 kinds of antibody comprising specific binding people Siglec-8, wherein this antibody comprises Fc district and is connected to The carbohydrate chain that the N-glucosides in this Fc district connects, the carbohydrate chain that wherein this N-glucosides less than 50% connects contains Fucosyl residues.

The carbohydrate chain that the compositions of 31. claim 30, the most substantially none this N-glucosides connect contains fucose Residue.

The compositions of 32. claim 30 or 31, wherein this antibody is humanized antibody, chimeric antibody or people's antibody.

The compositions of 33. any one of claim 30-32, wherein this antibody comprises variable region of heavy chain and variable region of light chain, wherein This variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises SEQ ID NO:62 The HVR-H2 of aminoacid sequence, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:63；And/or wherein should Variable region of light chain comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, and (ii) comprises SEQ ID NO:65's The HVR-L2 of aminoacid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:66.

The compositions of 34. claim 33, wherein this antibody comprises the aminoacid sequence selected from SEQ ID NO:2-10 Variable region of heavy chain；And/or comprise the variable region of light chain of aminoacid sequence selected from SEQ ID NO:16-22.

The compositions of 35. any one of claim 30-32, wherein this antibody comprises variable region of heavy chain and variable region of light chain, wherein This variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises SEQ ID NO:62 The HVR-H2 of aminoacid sequence, and (iii) comprise the HVR-H3 of the aminoacid sequence selected from SEQ ID NO:67-70；And/or Wherein this variable region of light chain comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, and (ii) comprises SEQ ID The HVR-L2 of the aminoacid sequence of NO:65, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:71.

The compositions of 36. claim 35, wherein this antibody comprises the aminoacid sequence selected from SEQ ID NO:11-14 Variable region of heavy chain；And/or comprise the variable region of light chain of aminoacid sequence selected from SEQ ID NO:23-24.

The compositions of 37. any one of claim 30-32, wherein this antibody comprises the amino selected from SEQ ID NO:2-14 The variable region of heavy chain of acid sequence；And/or comprise the variable region of light chain of aminoacid sequence selected from SEQ ID NO:16-24.

The compositions of 38. any one of claim 30-37, it comprises pharmaceutically acceptable supporting agent further.

The compositions of 39. any one of claim 30-38, the wherein binding affinity of this antibody on human Siglec-8 or affinity Higher to binding affinity or the affinity of people Siglec-8 than antibody 2E2 or 2C4.

The compositions of 40. any one of claim 30-39, wherein this antibody has at least about 70 DEG C in hot transfer assay method Tm。

The compositions of 41. claim 40, wherein this antibody has at least about 70 DEG C at least about 72 in hot transfer assay method DEG C Tm.

42. 1 kinds of antibody separated, it combines people Siglec-8 and kills the loose thin of expression Siglec-8 by ADCC activity Born of the same parents.

The antibody of 43. claim 42, wherein when administering therapeutic effective dose, this antibody cuts down expression Siglec-in experimenter The mastocyte of 8.

The antibody of 44. claim 43, wherein compared with baseline values before treatment, this antibody abatement obtains from this experimenter The mastocyte of the expression Siglec-8 of at least about 20% in sample.

The antibody of 45. claim 44, wherein this sample is tissue sample or biological fluid sample.

The antibody of 46. claim 45, wherein this tissue sample is one or more selected from lower group: skin, lung, bone marrow, and Nasal polyp.

The antibody of 47. claim 45, wherein this biological fluid sample is one or more selected from lower group: blood, a gas Pipe bronchoalveolar lavage fluid, and Nasal lavage fluid.

The antibody of 48. any one of claim 42-47, this antibody the most engineered is to improve antibody dependent cellular Cytotoxicity (ADCC) activity of mediation.

The antibody of 49. any one of claim 42-48, wherein the heavy chain at least one of this antibody or two are non-fucosylations 's.

The antibody of 50. claim 49, wherein this antibody is to have α 1, the cell that 6-fucosyltransferase (Fut8) knocks out System generates.

The antibody of 51. claim 49, wherein this antibody is at process LAN β Isosorbide-5-Nitrae-N-acetylglucosaminyltransferase (acetylglycosminyltransferase) cell line of III (GnT-III) generates.

The antibody of 52. claim 51, wherein this cell line other process LAN Gorky μ-mannosidase II (ManII).

The antibody of 53. claim 48, wherein this antibody comprises at least one place in Fc district and improves the aminoacid of ADCC activity and replace Generation.

The antibody of 54. any one of claim 42-53, wherein this antibody is humanized antibody, chimeric antibody or people's antibody.

The antibody of 55. any one of claim 42-54, wherein this antibody is human IgG1's antibody.

The antibody of 56. any one of claim 42-53, wherein this antibody is murine antibody.

The antibody of 57. any one of claim 42-56, wherein this antibody comprises:

A () variable region of heavy chain, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:88, and (ii) comprises SEQ The HVR-H2 of the aminoacid sequence of ID NO:91, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:94；With/ Or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:97, and (ii) comprises SEQ ID NO: The HVR-L2 of the aminoacid sequence of 100, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:103.

B () variable region of heavy chain, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:89, and (ii) comprises SEQ The HVR-H2 of the aminoacid sequence of ID NO:92, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:95；With/ Or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:98, and (ii) comprises SEQ ID NO: The HVR-L2 of the aminoacid sequence of 101, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:104.

C () variable region of heavy chain, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:90, and (ii) comprises SEQ The HVR-H2 of the aminoacid sequence of ID NO:93, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:96；With/ Or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:99, and (ii) comprises SEQ ID NO: The HVR-L2 of the aminoacid sequence of 102, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:105.

The antibody of 58. claim 57, wherein this antibody comprises:

A () comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:106；And/or comprise the amino of SEQ ID NO:109 The variable region of light chain of acid sequence.

B () comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:107；And/or comprise the amino of SEQ ID NO:110 The variable region of light chain of acid sequence.

C () comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:108；And/or comprise the amino of SEQ ID NO:111 The variable region of light chain of acid sequence.

The antibody of 59. any one of claim 42-56, wherein this antibody comprises variable region of heavy chain and variable region of light chain, wherein should Variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises SEQ ID NO:62's The HVR-H2 of aminoacid sequence, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:63；And/or wherein this is light Chain variable region comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, and (ii) comprises the ammonia of SEQ ID NO:65 The HVR-L2 of base acid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:66.

The antibody of 60. any one of claim 42-56, wherein this antibody comprises variable region of heavy chain and variable region of light chain, wherein should Variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises SEQ ID NO:62's The HVR-H2 of aminoacid sequence, and (iii) comprise the HVR-H3 of the aminoacid sequence selected from SEQ ID NO:67-70；And/or its In this variable region of light chain comprise the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, (ii) comprises SEQ ID NO: The HVR-L2 of the aminoacid sequence of 65, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:71.

61. 1 kinds of antibody, it combines people Siglec-8 and non-human primates Siglec-8.

The antibody of 62. claim 61, wherein this non-human primates is baboon.

The antibody of 63. claim 61 or 62, wherein this antibody comprises:

A () variable region of heavy chain, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:89, and (ii) comprises SEQ The HVR-H2 of the aminoacid sequence of ID NO:92, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:95；With/ Or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:98, and (ii) comprises SEQ ID NO: The HVR-L2 of the aminoacid sequence of 101, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:104.

B () variable region of heavy chain, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:90, and (ii) comprises SEQ The HVR-H2 of the aminoacid sequence of ID NO:93, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:96；With/ Or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:99, and (ii) comprises SEQ ID NO: The HVR-L2 of the aminoacid sequence of 102, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:105.

The antibody of 64. any one of claim 61-63, the wherein epi-position in this antibodies people Siglec-8 domain 1, wherein Domain 1 comprises the aminoacid sequence of SEQ ID NO:112.

The antibody of 65. any one of claim 61-63, the wherein epi-position in this antibodies people Siglec-8 domain 3, wherein Domain 3 comprises the aminoacid sequence of SEQ ID NO:114.

The antibody of 66. any one of claim 61-65, wherein this antibody is humanized antibody, chimeric antibody or people's antibody.

The antibody of 67. any one of claim 61-66, wherein this antibody is murine antibody.

The antibody of 68. any one of claim 61-67, wherein this antibody is IgG1 antibody.

The antibody of 69. any one of claim 43-60, wherein this experimenter behaves.

70. 1 kinds of nucleic acid, the antibody of its coding any one of claim 42-69.

71. 1 kinds of carriers, it comprises the nucleic acid of claim 70.

The carrier of 72. claim 71, it is expression vector.

73. 1 kinds of host cells, it comprises the nucleic acid of claim 70.

74. 1 kinds of methods generating antibody, it is included in the host cell cultivating claim 73 under conditions of generating this antibody.

The method of 75. claim 74, it farther includes to reclaim the antibody generated by this host cell.

The 76. anti-Siglec-8 antibody generated by the method for claim 74 or 75.

Treat in experimenter or prevent by the method for the cell-mediated disease expressing Siglec-8, the method bag for 77. 1 kinds Include claim 1-22 that this experimenter is used effective dose, the antibody of 28,42-69 and 76 any one or claim 30-41 The compositions of any one.

The method of 78. claim 77, wherein this disease is that eosinocyte (eosinophil) is disease mediated.

The method of 79. claim 77, wherein this disease is that mastocyte (mast cell) is disease mediated.

The method of 80. claim 77, wherein one or more symptoms of this antibody suppression atopic reaction.

The method of 81. claim 80, wherein this atopic reaction is I type hypersensitivity reaction.

The method of 82. claim 77, wherein this disease is selected from lower group: asthma, allergic rhinitis, nasal polyp, atopy skin Inflammation, chronic urticaria, Mastocytosis, eosinophilic leukemia, and eosinophilia's syndrome.

The method of 83. claim 77, wherein this disease is selected from lower group: few granulocytic asthma (pauci granulocytic Asthma), acute or chronic airways hypersensitivity, eosinophilic esophagitis, Qiu-execute Er Shi (Churg-Strauss) combines Simulator sickness, the inflammation relevant with cytokine, the inflammation relevant with the cell expressing Siglec-8, with the cell expressing Siglec-8 Relevant is pernicious, physical urticaria, cold urticaria, pressure urticaria, bullous pemphigoid, food anaphylaxis, and Allergic bronchopulmonary aspergillosis (ABPA).

The method of 84. any one of claim 74-83, wherein this experimenter suffers from by suction-type corticosteroid, fugitive β 2 Agonist, long-acting β2agonists, or the asthma that a combination thereof is the most appropriately controlled.

85. 1 kinds of methods that abatement expresses the mastocyte of Siglec-8 in experimenter, it includes having used this experimenter The antibody of the combination people Siglec-8 of effect amount, wherein this antibody kills the mastocyte expressing Siglec-8 by ADCC activity.

The method of 86. claim 85, wherein compared with baseline values before treatment, this antibody abatement obtains from this experimenter The mastocyte of the expression Siglec-8 of at least about 20% in sample.

The method of 87. claim 86, wherein this sample is tissue sample or biological fluid sample.

The method of 88. claim 87, wherein this tissue sample is one or more selected from lower group: skin, lung, bone marrow, and Nasal polyp.

The method of 89. claim 87, wherein this biological fluid sample is one or more selected from lower group: blood, a gas Pipe bronchoalveolar lavage fluid, and Nasal lavage fluid.

The method of 90. any one of claim 85-89, this antibody the most engineered is to improve antibody dependent cellular Cytotoxicity (ADCC) activity of mediation.

The method of 91. any one of claim 85-90, wherein the heavy chain at least one of this antibody or two are non-fucosylations 's.

The method of 92. claim 91, wherein this antibody is to have a α 1, and it is thin that 6-fucosyltransferase (Fut8) knocks out Born of the same parents system generates.

The method of 93. claim 91, wherein this antibody is at process LAN β Isosorbide-5-Nitrae-Kre2GnTI II (GnT- III) cell line generates.

The method of 94. claim 93, wherein this cell line other process LAN Gorky μ-mannosidase II (ManII).

The method of 95. claim 90, wherein this antibody comprises at least one place in Fc district and improves the aminoacid of ADCC activity and replace Generation.

The method of 96. any one of claim 85-95, wherein this antibody is humanized antibody, chimeric antibody or people's antibody.

The method of 97. any one of claim 85-96, wherein this antibody is human IgG1's antibody.

The method of 98. any one of claim 85-97, wherein this antibody comprises:

A () variable region of heavy chain, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:88, and (ii) comprises SEQ The HVR-H2 of the aminoacid sequence of ID NO:91, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:94；With/ Or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:97, and (ii) comprises SEQ ID NO: The HVR-L2 of the aminoacid sequence of 100, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:103.

B () variable region of heavy chain, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:89, and (ii) comprises SEQ The HVR-H2 of the aminoacid sequence of ID NO:92, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:95；With/ Or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:98, and (ii) comprises SEQ ID NO: The HVR-L2 of the aminoacid sequence of 10

C () variable region of heavy chain, it comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:90, and (ii) comprises SEQ The HVR-H2 of the aminoacid sequence of ID NO:93, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:96；With/ Or variable region of light chain, it comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:99, and (ii) comprises SEQ ID NO: The HVR-L2 of the aminoacid sequence of 102, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:105.

The method of 99. claim 98, wherein this antibody comprises:

A () comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:106；And/or comprise the amino of SEQ ID NO:109 The variable region of light chain of acid sequence.

B () comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:107；And/or comprise the amino of SEQ ID NO:110 The variable region of light chain of acid sequence.

C () comprises the variable region of heavy chain of the aminoacid sequence of SEQ ID NO:108；And/or comprise the amino of SEQ ID NO:111 The variable region of light chain of acid sequence.

100. the method for any one of claim 85-97, wherein this antibody comprises variable region of heavy chain and variable region of light chain, wherein should Variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises SEQ ID NO:62's The HVR-H2 of aminoacid sequence, and (iii) comprise the HVR-H3 of aminoacid sequence of SEQ ID NO:63；And/or wherein this is light Chain variable region comprises the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, and (ii) comprises the ammonia of SEQ ID NO:65 The HVR-L2 of base acid sequence, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:66.

The method of 101. any one of claim 85-97, wherein this antibody comprises variable region of heavy chain and variable region of light chain, wherein should Variable region of heavy chain comprises the HVR-H1 that (i) comprises the aminoacid sequence of SEQ ID NO:61, and (ii) comprises SEQ ID NO:62's The HVR-H2 of aminoacid sequence, and (iii) comprise the HVR-H3 of the aminoacid sequence selected from SEQ ID NO:67-70；And/or its In this variable region of light chain comprise the HVR-L1 that (i) comprises the aminoacid sequence of SEQ ID NO:64, (ii) comprises SEQ ID NO: The HVR-L2 of the aminoacid sequence of 65, and (iii) comprise the HVR-L3 of aminoacid sequence of SEQ ID NO:71.

The method of 102. any one of claim 85-101, wherein this experimenter has by expressing the cell-mediated of Siglec-8 Disease.

The method of 103. any one of claim 85-102, wherein one or more symptoms of this antibody suppression atopic reaction.

The method of 104. claim 103, wherein this atopic reaction is I type hypersensitivity reaction.

The method of 105. claim 102, wherein this disease is selected from lower group: asthma, allergic rhinitis, nasal polyp, atopy Dermatitis, chronic urticaria, Mastocytosis, eosinophilic leukemia, and eosinophilia's syndrome.

The method of 106. claim 102, wherein this disease is selected from lower group: few granulocytic asthma, and acute or chronic air flue surpasses Sensitivity, eosinophilic esophagitis, churg-Strauss syndrome, the inflammation relevant with cytokine, with expression The inflammation that the cell of Siglec-8 is relevant, relevant with the cell expressing Siglec-8 is pernicious, physical urticaria, col nettle Measles, pressure urticaria, bullous pemphigoid, food anaphylaxis, and allergic bronchopulmonary aspergillosis (ABPA).

The method of 107. any one of claim 85-106, wherein this experimenter suffers from by suction-type corticosteroid, fugitive β 2 agonist, long-acting β2agonists, or the asthma that a combination thereof is the most appropriately controlled.

108. 1 kinds of pharmaceutical compositions, it comprises claim 42-69 and the antibody of 76 any one and pharmaceutically acceptable supporting agent.

109. 1 kinds of test kits, it comprises claim 1-22, the antibody of 28,42-69 and 76 any one or claim 30-41 The compositions of any one.